Microbiota in colorectal cancer related to liver metastasis

Abstract

The prevalence of colorectal cancer (CRC) is increasing annually and metastasis is the principal cause of death in patients with CRC, with the liver being the most frequently affected site. Many studies have shown a strong interplay between the gut flora, particularly Fusobacterium nucleatum (F. nucleatum), Escherichia coli, and Bacteroides fragilis, and the development of gut tumors. Some strains can induce gut inflammation and produce toxins that directly harm gut epithelial cells, ultimately accelerating the onset and progression of CRC. However, little clinical evidence exists on the specific interplay between the gut microflora and colorectal cancer liver metastasis (CRLM). Some research showed the existence of viable F. nucleatum in distant metastasis of CRC. Subsequently, gut microbiota products, such as lipopolysaccharides, sodium butyrate, and protein cathepsin K, were also found to affect the development of CRC. This article summarizes the mechanism and research status of the interplay between gut microflora and CRLM, discusses the importance of gut microflora in the treatment of CRLM, and proposes a new approach to understanding the mechanism of CRLM and potential treatments for the microbiome. It is anticipated that the gut microbiota will be a formidable therapeutic and prophylactic tool for treating and preventing CRLM.

Background

Colorectal cancer (CRC) is a universal malignant neoplasm that accounts for 10% of the global cancer diagnoses and fatalities annually (1,2), and its occurrence rate is increasing annually. It is estimated that by the 2040s, the proportion of patients with CRC will substantially rise to 25 million (3,4). The causes of CRC are multifactorial. In particular, older adults are at risk of CRC. Additionally, unhealthy eating habits combined with alcohol abuse, fat consumption, insufficient physical activity, and smoking can increase the likelihood of CRC (5). CRC originates mainly from dysplastic adenomatous polyps (6), which originate from abnormal crypts and evolve into neoplastic precursor lesions, namely, polyps, eventually developing into CRC within an estimated 15 years. This process requires a series of steps, including the deactivation of several tumor suppressor genes and activation of proto-oncogenes, which can cause the normal epithelium to develop into precancerous adenomatous polyps, causing it to grow faster and eventually lead to local invasion and metastasis. CRC cells are thought to gradually develop from stem cells or stem-like cells, which can not only deactivate tumor suppressor genes and activate oncogenes, but also give rise to genetic and epigenetic modifications, eventually leading to recurrence and treatment failure in patients with cancer (7). Approximately 25% of patients with CRC are thought to have distant metastases at their first hospital visit, which is the primary cause of their ultimate death, and approximately 50% develop metastases as the disease progresses (8). Metastasis most commonly occurs in the liver, followed by the lungs (9).

Considerable research has confirmed the vital role of gut microbial dysbiosis in CRC development (10-13). Some pathogenic bacteria trigger chronic inflammation, thereby promoting the development of CRC (14,15). Furthermore, gut microbiota can interact with cancer cells to affect the tumor microenvironment, thereby augmenting neoplasm invasiveness (16,17) and promoting distant metastasis of invasive CRC in an epithelial-mesenchymal transition-susceptible tissue environment through a synergistic inflammatory tumor-promoting mechanism (18). Consequently, a profound analysis of the relationship between gut microbiota, CRC and its liver metastasis will surely provide insights into the mechanisms of colorectal cancer liver metastasis (CRLM).

Gut microbiota

Tumors were thought to exist in sterile environments; however, advances in sequencing technology have changed this view and fueled an increase in intratumoral microbiome research. Studies have demonstrated (19-21) that most human cancer types have intratumoral microbiota, including bacterial communities located around and deep within tumor tissues. Based on some intrinsic characteristics of tumor tissues, such as leaky vasculature, hypoxia, necrotic tissue, and immune privilege (22), tumor lesions may support bacterial invasion, survival, and growth. As the most colonized part of the digestive tract, the colorectum includes an abundance of various microorganisms that are closely linked to host intestinal epithelial cells (23). A representative gut microbiome may comprise billions of different types of microbial cells, more than three million genes (24), and may account for 70% of the human microbiome (25). The gut system can contribute to cell carcinogenesis and play a key role in numerous human diseases (26). Approximately 20% of neoplasms are associated with microbiota that regularly colonizes the gut (27). Although gut microbiota is known to have a marked effect on the occurrence and progression of CRC and new evidence suggests that it also affects CRC metastasis (28), the underlying mechanisms remain unclear.

Main flora related to CRC metastasis

In previous systematic reviews, Fusobacterium nucleatum (F. nucleatum) was found to have the highest proportion in the intestines of healthy individuals and patients with CRC, although it was not the most dominant intestinal organism. Additionally, enterotoxigenic F. nucleatum, Escherichia coli (E. coli), Bacteroides fragilis (B. fragilis), Bacillus, and Salmonella species have been shown to be candidate pathogens for CRC progression (29,30).

E. coli is a gram-negative and symbiotic bacterium in the gut. Some E. coli strains can give rise to gut inflammation and generate toxins with carcinogenic latent capacity, which can interact with gut epithelial cells to trigger DNA double-strand breakage and chromosomal instability, ultimately accelerating the incidence and development of CRC (31). E. coli carries a conserved gene island, which can produce a genotoxic substance called polyacetyl ethyl alcohol that can cause DNA damage and lead to cancer (32). Swidsinski et al. (33) used a classic gentamicin protection test to confirm the colonization of E. coli in the gut mucosa. The detection rate of E. coli in the tumor samples was 81%, whereas no E. coli was detected in the control group, confirming that E. coli has a strong association with tumor samples.

F. nucleatum, an anaerobic gram-negative bacterium, primarily colonizes the mouth and colon. Proteins on the extracellular surface can have specific interactions with the complementary structures on the surface of the host cell, bridge a variety of bacteria in the biofilm through adhesins, and generate important lipopolysaccharides (LPS), endotoxins, and hemolysins, making them more toxic than most normal anaerobic bacteria. In addition, this bacterium easily spreads, sometimes causing the destruction of tumors, polyps, and other harmless tissue blocks. Even changes in the local inflammatory environment may lead to excessive growth of nonfunctional tissues; hence, it is also called “oncobacterium.” Several studies have shown that F. nucleatum affects the occurrence and metastasis of CRC (17,34,35). Unbiased genomic analyses have shown that F. nucleatum is richer in human colon cancers and adenomas than in non-cancerous colon tissues (36,37), which has been confirmed in studies of multiple cohorts of patients with colon cancer worldwide (38,39). Various mechanisms, including signal transduction pathways and inflammation, play key roles in the occurrence and development of CRC (40). F. nucleatum is abundant in 60% of proximal CRC metastatic lymph nodes (41). Furthermore, F. nucleatum stimulates the expression of cytokines that subsequently interact with immunocytes to increase the incidence of CRC (42). Borges-Canha et al. (40) reported that, compared with those from mice fed with Streptococcus, strains isolated from F. nucleatum-fed Apc Min/+ mice had a faster development of CRC. Therefore, colon cancer tumor stage, metastasis, chemotherapy resistance, sex, and prognosis can be determined by measuring F. nucleatum DNA levels in clinical practice.

B. fragilis is an obligate anaerobic gram-negative bacterium and an opportunistic pathogen. It manifests primarily as an internal infection and frequently exhibits parasitic behavior within the oral cavity, intestines, and female vagina. B. fragilis includes two subtypes called non-enterotoxigenic B. fragilis (NTBF) and enterotoxigenic B. fragilis (ETBF). The latter can markedly affect the occurrence and growth of CRC. ETBFs not only directly release toxins to destroy gut epithelial cells, but also promote the splitting of tumor suppressor proteins and even destroy the gut mucosa. In 26 studies (43) in which mice were administered ETBF or NTBF, an important relationship was found between B. fragilis and C-reactive protein, which is effective in treating acute inflammation and affects the occurrence of tumors.

Pathogenesis of tumor metastasis

Previous studies have shown that neoplastic cell migration is a dynamic, complex, and multimechanistic procedure. In 1889, Paget, a British surgeon, put forth the groundbreaking “seed and soil” theory (44) to suggest that neoplasm cells can cause metastasis when they interact with a specific organ microenvironment. He found that the result of metastasis is not accidental; some tumor cells (“seeds”) exhibit specificity. That is, the cancerization of normal cells requires tumor over-proliferation to provide nutrients, enter the circulation, colonize the metastatic focus, and continue to grow (45,46). This pioneering and classic “seed and soil” theory to explain distant metastasis of malignant tumors has been continuously verified and developed for more than a hundred years and has been supported by ever increasing research, suggesting that the occurrence of distant metastasis of tumors requires more than just tumor cells. Metastatic target organs must also provide a metastatic microenvironment to enhance the potential of cells to colonize distant areas. Exploring the mechanism of CRLM must start with two aspects: CRC cells and the liver microenvironment. A balanced and homeostatic liver microenvironment favors the metastasis of CRC cells. CRC cells can influence the metastasis microenvironment by secreting a variety of cytokines or releasing substances, such as those containing DNA fragments, and proteins, and form a pre-metastatic niche, providing suitable “soil” for CRC tumor cells to transfer and colonize the liver.

The effect of gut microbiota on CRC cells can be shown to promote cell carcinogenesis by influencing inflammation, immune response and destruction of DNA. An imbalance in gut microbiota can produce carcinogenic secondary metabolites, regulate the gut immune response, mediate the occurrence of an inflammatory state, cause the overexpression of cytokines and chemokines, and degrade the intestinal epithelium. Cancerization will be stimulated in the gut cells, if the cells are always in an inflammatory microenvironment. Wong et al. (11) administered stool samples from patients with CRC and found that both inflammatory markers in mice and the ratio of helper T cells in the gut increased, which can play a key role in the occurrence of cancer in mice bred and raised in sterile environments. Besides, some microbial metabolites have been confirmed to cause DNA destruction (12), leading to a sustained inflammatory response and ultimately causing DNA damage and promoting the development of cell carcinogenesis. Furthermore, the gut microbiota affects the formation of intestinal bacterial biofilms. Gut bacterial biofilms are aggregates formed by the organized growth of various gut bacteria that can improve host defense. Once the balance of the microflora is disrupted, the host is vulnerable to damage, and the cells are prone to transformation or cancerization.

The gut microbiota needs to act on not only tumor cells but also the microenvironment to have an impact on metastasis. A balanced and homeostatic liver microenvironment favors the metastasis of CRC cells. Cao et al. (47) reviewed the formation mechanism, composition characteristics, and process of promotion of tumor metastasis in the pre-tumor metastasis microenvironment and proposed, for the first time, six major characteristics of the pre-tumor metastasis microenvironment, which can enable tumor cells to colonize and promote metastasis. In addition to neoplasm cells, the tumor microenvironment also consists of immunocytes, tumor blood vessels, tumor-associated macrophages, tumor-associated fibroblasts, the extracellular matrix, and other cells (48), which have a major impact on neoplasm development. Therefore, the gut microbiota can affect the liver microenvironment by affecting immunosuppression and inflammation, which can colonize tumor cells and promote metastasis. Studies have shown (49) that gut microbiota can mediate the movement of cancer cells from the intestine to the liver by destroying the intestinal mucosa. Changes in gut microbiota increase intestinal permeability, translocate endotoxins, and allow hepatotoxins to enter the liver (50). Furthermore, Previous reviews have shown that a microenvironment conducive to tumor growth can form through the intestinal microflora and accelerate the progression and metastasis of tumor cells (51). For instance, LPS can accelerate the epithelial-mesenchymal transformation of primary sites to cause a deficiency in the inner mucosal layer and damage the mucosal barrier, encouraging microbiota spread by affecting changes in the intestinal microenvironment, to enhance permeability (49). As a crucial component of gram-negative bacteria, it is universally acknowledged that LPS causes liver metastasis by stimulating inflammation (52,53). LPS, an immunostimulatory ligand, activates many pathways that promote CRC metastasis. LPS can stimulate the TLR4 receptor as well as increase β1 integrin-mediated cell adhesion (31,54,55). Metastasis is a multi-gene, multi-step, complex process involving close cooperation between neoplastic cells, immunity, and inflammation. The liver, which is the largest visceral part of the body, has a distinctive structure that allows it to perform multiple functions, including protein synthesis and portal blood detoxification. The liver is the most commonly affected site when CRC metastasizes (56,57). Pathologically, the liver receives nearly three-quarters of its blood supply from intestinal veins through the portal vein. Researchers have suggested that both CRC cells and gut microbiota enter the liver and reproduce via the portal vein (51). Intestinal mucosal lymphocytes also migrate between the two organs to defend against pathogens. The retrospective data of Weiss (58) and Sugarbaker (59) suggested that anatomical or mechanical factors may lead to regional metastasis. For instance, the colonization of symbiotic bacteria to the liver follows the metastasis of cancer cells through the bloodstream (60). However, the metastasis of various cancers to distant organs is site-specific. Findings from an experiment using mice showed that neoplastic cells may linger in extremely fine blood vessels at the beginning of migration; however, further progress is regulated by cells of specific organs (61).

Cancer metastasis involves several interrelated steps. Treatment of CRLM should target both CRC cells and other host factors, such as the tumor microenvironment, that may promote these CRC cells to reproduce and cause metastasis to other tissues or, in some cases, organs (62).

Research status of CRLM

Although many articles on the gut microbiota and CRC have been published, little clinical evidence exists on the specific interplay between the gut microflora and CRLM. Findings in 2017 confirmed the existence of viable F. nucleatum in distant metastasis of CRC (16). According to this study, F. nucleatum and commensal bacteria, such as Bacteroides, migrate to the liver along with the metastasis of cancer cells. In this study, F. nucleatum exhibited increased abundance in both primary and metastatic lesions in CRC, compared with that in hepatocellular carcinoma, suggesting that F. nucleatum and symbiotic bacteria cause the development, diffusion, and migration of CRC cells. The identification of nearly identical live F. nucleatum strains in matched primary and metastatic CRCs confirmed the persistence of live F. nucleatum during the metastatic process and suggested that F. nucleatum may colonize metastatic areas along with CRC cells. This study provides a solid theoretical approach for the use of flora to treat cancer metastasis (16).

As a key component of gram-negative bacteria, the substantial role of LPS on liver metastasis has been widely reported. In a 2018 study, downregulated LPS, which are products of the gut microbiota, promoted CRC immunotherapy and alleviated liver metastasis (63). An LPS-targeting fusion protein that selectively expresses LPS capture proteins and blocks LPS in tumors to promote tumor treatment was also designed and engineered in the study, indicating LPS as a key therapeutic target for CRLM.

In a 2019 study, the gut microflora influenced CRC metastasis through the secreted protein cathepsin K (31). After treating the mice with antibiotics, MC38 cells, which are colon cancer cells in mice, were seeded into the guts of the mice, and E. coli was subsequently added to affect the balance of the gut microflora. The results showed that liver metastasis and cathepsin K overexpression occurred in the intervention group, demonstrating that bacterial imbalance promotes CRLM.

In 2020, Ma et al. (64) used sodium butyrate (NaB) — the main gut microbial flora fermentation product — instead of LPS in a mouse model to modulate CRLM. The findings indicated that NaB could reduce liver metastasis, thereby ameliorating the disruption of CRC in mice with liver metastasis, which in turn improved the gut microbial cluster and immune response of mice. The results of this study indicated that NaB may be effective for treating CRLM (64) and gut microorganisms may be new targets for liver metastasis therapy in gut cancer.

Earlier literature confirmed the existence of gut microbiota in metastases of CRC, but provided no direct evidence that gut microbiota is involved in CRC metastasis. In a 2021 study (50) the most common liver metastasis originating from the gut was related to the spread of colonized E. coli from the tumor to the liver. Additionally, a specific E. coli C17 strain was found to induce gut vascular barrier damage, causing the gut microbiota to pass through the barrier to reach the liver. E. coli C17 was capable of causing gut vascular barrier damage, which allows the gut microbiota to breach the barrier and penetrate the liver. Moreover, cancer cells were stronger at sites where E. coli C17 accumulated. The high PV-1 also indicated that the intestinal vascular barrier was damaged, and the permeability was substantially increased, which means that the gut microbiota was more likely to penetrate the barrier for dissemination to provide a good “soil” for hepatic metastasis.

In 2022, researchers explored the link between Fusobacterium and CRLM. Some researchers have concluded that changes in the Proteus mirabilis and common Bacteroides ratio affect CRLM, which is likely influenced by hepatic Kupffer cells (65). Additionally, more studies have been conducted on F. nucleatum than on other bacterial species. This bacterium diminishes METTL3-mediated m⁶A alteration, aids in CRC metastasis (17), and stimulates ALPK1/NF-κB (34), suggesting that F. nucleatum can increase the diffusivity of CRC cells.

In 2023, Zheng et al. (66) summarized the effects and molecular mechanisms of microflora in the metastatic process of CRC. The presence of F. nucleatum, B. fragilis, and E. coli has been linked to the spread of CRC to distant areas, suggesting that targeting intestinal bacteria may be a viable way to suppress CRLM.

Conclusions

Recent studies have shown that the gut microbiota has unique advantages for CRC-related inflammation, prevention, immune regulation, and tumor invasion and proliferation. Overall, a strong interplay between the gut flora, particularly F. nucleatum, E. coli, and B. fragilis, and the development of gut tumors was established in this review, based on findings from the discussed studies. This review offers new perspectives and a challenging research topic on the prevention and treatment of CRC by regulating the gut flora; however, the role of gut microbiota in metastasis has not been fully determined, which is an immense hindrance to successful CRC treatment. The existence of intratumoral bacteria has been confirmed previously, the idea that the flora can migrate to the liver along with the metastasis of cancer cells was indicated, and the effect of the flora on CRLM was confirmed by the analysis of bacterial body components and secreted proteins. However, most current research are experimental CRC models, and corresponding clinical trials still lack corresponding clinical trials. Concurrently, owing to organizational differences, technical difficulties, neoplasm staging, and cross-species transformation, problems remain in clinical practice and many challenges need to be overcome.

Consequently, microflora is a powerful target for the treatment of patients with CRLM. A detailed analysis of the relationship between gut microbiota, CRC, and liver metastasis will provide insights regarding the mechanisms of CRLM. In terms of therapeutic strategies, it is necessary to consider not only the signaling regulatory pathways of gut microbiota on CRLM, but also the effects of live and dead bacteria, bacterial composition, secreted proteins, and different bacterial counts. In the future, we should focus on exploring new ideas for the prevention, early intervention and treatment of CRLM by intervening in the microflora of antibiotics or blocking the production of bacterial components. It is anticipated that the gut microbiota will be a formidable tool in the treatment against CRLM and will provide new strategies for preventing CRLM in the coming years.