Understanding the role of the gut microbiome in gastrointestinal cancer: A review

1“Microbiome” and “cancer” are the most popular topics of today’s modern age. All human surfaces and all the cavities that communicate with the outside are populated by an ecosystem of microorganisms, such as bacteria, viruses, protozoa, fungi, and archaea. This population of microbes has a complex, individual, and variable nature; its composition is influenced by the genetics of the host, dietary habits, lifestyle, and microbial exposure at birth. An average of ten times more bacteria has been calculated than the number of cells in the human body, between 500 and 1,000 different species of microorganisms. The scientific community has agreed on defining “microbiota” as the totality of microbial organisms present in certain environments, while the term “microbiome” the genetic information inherent in the microbiota itself. The microbiome presents a great variability in its composition both between different subjects and within the same subject, in different anatomical sites and tissues. Individual and inter-individual differences are thought to be the underlying cause of many diseases and health problems, in gastrointestinal (GI) cancers; therefore, they play a key role in both diagnosis and treatment.

2Although cancer is generally accepted as a disease caused by genetic characteristics and environmental factors, it has been reported that the effect of microorganisms on cancer could be approximately 20% of cancers human beings suffer from. Digestive system cancers account for approximately one-third of cancers that result in death. At least 15%–20% of cancers are triggered by infectious agents; 20%–30% are associated with tobacco products, 30%–35% with diet, lack of physical activity, and/or energy balance disorder (obesity). Targeted therapies are effective in the treatment of certain types of cancer. Many cancer types are treated by traditional chemotherapy treatments with varying efficacy and side effects. Cancer prevention is the main target, and it is reported that a better understanding of the interactions between gut microbiota, barrier function, and inflammatory responses is significant in identifying new targets in cancer therapy.

3This review aims to examine the role of the gut microbiota in the development of digestive system cancers, point out the potential factors-metabolites that can modulate this relationship, and comprehensively evaluate their potential role in diagnosis and treatment.

4Despite the heterogeneity found in the microbiome, based on the analysis of deoxyribonucleic acid (DNA) sequences from 39 samples belonging to subjects of six different nationalities, three main bacterial strains, which are Bacteroides, Prevotella, and Ruminococcus have been identified which are independent of age, sex, geographical area or diet, and based on the prevalence of one of them, each individual is cataloged in one of three enterotypes. This enterotype is linked to specific functions of the subject, such as the production of some nutrients like vitamins and the predisposition to some non-communicable diseases (NCDs).

5The number of microorganisms is very high in the oral cavity, where it consists mainly of Firmicutes and Proteobacteria. At the level of the esophagus, the Firmicutes increase even more. In the stomach involved in the secretion of hydrochloric acid in which the pH is strongly acidic (pH ≈ 2), there are a few bacteria among which the most abundant are the Firmicutes, Proteobacteria, and Actinobacteria. The duodenum also has a low microbial population due to the rapid transit time and the secretion of bile and pancreatic fluids that kill most of the ingested bacteria, and because of propulsive motor activity that prevents stable colonization of the lumen.

6There is a progressive increase in the number of species from mouth to ileum: first, the Gram (−) and then the obligate anaerobes increase. At the level of mouth and ileum, in which the digestion and absorption of monosaccharides, amino acids, and fatty acids take place, there are Enterococci and Lactobacilli, while in the ileum and the colon (at the level of which the bile acid is absorbed) there is a myriad of bacteria, including Ruminococcus, Staphylococcus, Streptococcus, Peptococcus, Escherichia, Eubacterium, Clostridium, and many others are less abundant such as Proteobacteria, Actinobacteria and Fusobacteria. The gut microbiome is the set of symbiotic microorganisms, which are bacteria, viruses, fungi, and protozoa, found in the digestive tract of mankind, about 1010-1012cells/Gram or milliliter of luminal content, 500–1,000 different species, mostly anaerobic non-sporogenous, and species that cannot be identified by conventional methods.

7Most of the species that make up the gut microbiome of the gastrointestinal tract (GIT) are included in the five main phyla in the taxonomic classification. They are listed as Firmicutes (60%–80%), Bacteroidetes (15%–25%), Actinobacteria (2.5%–5%), Proteobacteria (1%–10%) and Verrucomicrobia (0.1%–2.2%). The main bacterial phyla and species in the human digestive tract microbiota are shown in Table 1.

8There is fast-growing literature examining and reporting the relationship between the bacterial composition, structure, and functional capacity of the microbiome in health and diseases. In the gut microbiome, a decrease in beneficial bacteria populations and an increase in pathogenic bacteria populations, changes in bacterial content, and deterioration of balance are defined as dysbiosis. Dysbiosis affects health negatively by preventing the regular functioning of the immune system and may play a role in the pathogenesis of some diseases. Dysbiosis has been correlated with inflammatory diseases, metabolic disorders, and tumors.

9The lack of adequate laboratory techniques has for a long time conditioned the study of the intestinal microbiome, being the classical inadequate cultivation method since most of the flora is made up of anaerobic germs. Fortunately, the development of techniques based on the sequencing of the 16S ribosomal RNA (16S rRNA) has facilitated the identification and classification of bacteria.

10The colonization of the gastrointestinal tract begins at birth with Bacteroides and Bifidobacteria in the event of a vaginal delivery. On the other hand, it mostly begins with feeding in the cesarean delivery.

11In addition to the type of birth, the type of feeding is also a significant determinant of the intestinal microbiota of infants. While Bifidobacterium and Lactobacillus provide colonization in the intestinal microbiota of breastfed infants; Enterococcus, Enterobacteria, Bacteroides, Clostridium, and other anaerobic Streptococcus bacteria provide colonization in the intestinal microbiota of formula-fed infants. Microbiome composition and diversity may vary depending on age. The first year of life is fundamental for the establishment of the microbiome, which then variably evolves into adulthood from person to person. In children between 1 and 7 years of age, there is a greater number of Enterobacteria than in adults with a greater abundance of general Bifidobacterium and Clostridium. The composition of the microbiota is very dynamic in the first 3 years of life and then becomes more stable and complex in adulthood. In geriatric age, there is a significant variation in its composition with an increase in Bacteroides, Escherichia coli, Streptococcus, Clostridia, Lactobacilli, and Bifidobacterium decrease.

12The microbiome is particularly abundant in the cecum and the right colon due to the large availability of substrates and the favorable environment for bacterial growth (low transit time, ready availability of nutrients, favorable pH, etc.). The stomach has a bacterial concentration of about 107 bacteria, therefore much lower than the distal tracts of the gastrointestinal system. The duodenum bacteria have been little studied, except for their involvement in some diseases, such as irritable bowel syndrome (IBS), in which both bacterial growth and a reduction in microbial diversity have been found. In any case, tumors of the duodenum are rare, and the relevance of bacterial involvement has not yet been investigated extensively. Besides, although the jejunum and ileum are the longest part of the gastrointestinal system and with the widest contact surface, the recovery of samples in this region remains a challenge. Molecular analyses of the 16S rRNA gene have shown that jejunum hosts a bacterial community less complex than the distal ileum and the large intestine; nevertheless, the knowledge on the microbial community of the small intestine is still limited both in healthy individuals and patients. On the other hand, the colon hosts one of the most populous and diverse bacterial communities that are distributed between the lumen and the mucosa.

13Dietary patterns play a significant role in the modulation of the gut microbiome. Accumulated literature investigating the roles of nutrition in the modulation of the microbiome. Nutrition has the potential to affect gut microbiota composition and function by influencing microbial diversity, microbial taxonomy, gene expressions, and enzyme activities. Macronutrient and phytochemical content of the diet; some specific foods (probiotics, fermented foods, whole grains, oilseeds, and fruits, etc.) and the overall diet have the potential to modulate the gut microbiome in a short time. In one of the first intervention studies examining the effects of short- and long-term dietary habits on the microbiome, it was shown that long-term dietary habits were associated with three enterotypes (Bacteroides, Prevotella, and Ruminoccous) determined through dominant groups. Bacteroides enterotypes were defined in diets rich in animal protein and fat, and Prevotella enterotypes in diets rich in complex carbohydrates. At the same time, in this study, it was shown that the change in gut microbiome started within 24 h of short-term dietary intervention but did not cause significant changes to affect enterotypes. In another dietary intervention study, butyrate production increased when the fiber content of the diet was increased; the fat content decreased for 2 weeks; and secondary bile acid synthesis, colonic mucosal proliferation, and inflammation have been shown to decrease. For example, the gut microbiome of individuals who are carnivores or vegetarians differs. Changes in diet cause changes in the number and content of microorganisms in the microbiome. It was found that the ratios of Prevotella, Lactobacillus, and Bifidobacterium bacteria and fecal short-chain fatty acid (SCFA) levels were high in the microbiota of individuals with high adherence to the Mediterranean diet (MedDiet), and Clostridium species are seen to be lower.

14Interestingly, the diet influences the composition of the gut microbiome especially if the new eating habits last over time. A study suggests that low-fiber diet cause some reversible microbiota changes in mice, but the diversity of the microbiota is decreased if the diet continues for several generations. It is also noted that a modern diet low in fiber contributes to the loss of taxa over generations and may be responsible for the lower diversity microbiota observed in the industrialized world.

15There is also a high interindividual and geographical diversity. Nutritional habits are also reported to play a significant role in this diversity. The study of African children showed a greater presence of Prevotella, Xylanibacter, and Treponema, which possess enzymes to maximize the extraction of energy from xylans and cellulose, present in large quantities in the low-calorie but a high-fiber diet of these children. In studies conducted in adult groups, the diets of those living in rural areas were based on plant-based foods, the diversity in microbial composition was high and Prevotella was dominant; on the other hand, it has been found that microbial diversity is less and Bacteroides are dominant in those living in developed societies that consume foods rich in animal foods and processed products. All these pieces of evidence shed light on the need for well-designed studies that will evaluate the nutritional microbiota relationship from different aspects to develop correct nutritional recommendations for the regulation of microbiota.

16As well as diet, even antibiotic treatments, invasive pathogens, drug-taking, or stress can disturb the balance of organisms and cause dysbiosis. It shows that antibiotic therapy in the first years of life increases the risk of inflammatory bowel disease. A study showed that the microbiota is modified by the administration of antibiotic therapy. It has been known for a long time that excessive use of antibiotics has some negative effects on increasing antibiotic-resistant pathogens.

17All these factors, individually or in combination, affect the intestinal microbiome, and alteration of the intestinal microbiome is related to various diseases including obesity, type 2 diabetes, atherosclerosis, cancer neurological and psychiatric diseases, inflammatory bowel diseases (IBD), and autoimmune diseases. Hence, the human microbiome including the GI tract represents a real organ adapted to protect our life and our wellbeing but is also willing to be altered in response to any factors.

18The intestinal flora has a metabolic, structural, and protective role; carries out functions such as metabolizing the indigestible components of the diet, producing vitamins, contributing to the fortification of the mucosal barrier, and preventing the increase of pathogenic microorganisms. Therefore, the microbiota plays an important role in the development and regulation of the immune system. There is evidence in favor of the role of the microbiota in the development of B lymphocytes and the regulation of T helper lymphocytes. Several studies in the mouse have shown that tolerogenic dendritic cells and regulatory T cells (Tregs) are absent in germ-free mice and that both commensal bacteria and their metabolites, such as butyric acid, are necessary for the development of Tregs.

19The gut microbiome also has many roles in the digestion and metabolism of nutrients. The intestinal flora, influencing the metabolism with specific enzymes, increases the extraction of energy and therefore influences the control of body weight. Resident bacteria are a fundamental line of resistance to colonization by exogenous microbes, reducing the availability of nutrients to potential pathogens, competing for sites of attack on the brush border of intestinal epithelial cells, and producing some antimicrobial substances. Besides, among the components of the human microbiota are listed those that cause fermentation (80%) such as Lactobacillus and Bifidobacteria, and those that cause putrefaction of the remains (20%) such as Escherichia, Bacteroides, Eubacteria, and Clostridium. The fermentation of non-digestible food residues and endogenous mucus produced by the epithelium is the main source of energy in the colon. The fibers are resistant to digestion in the small intestine due to the conformation of the glycosidic bond: the enzymes of the human small intestine can only split α-type bonds, but not those of the β type. This explains why amylose, a glucose polymer with α-1,4 glycosidic bond, is digestible, while cellulose, a glucose polymer with β-1,4 glycosidic bond, is indigestible due to the lack of the specific enzyme. The distinctive feature of the carbohydrate type is the difference in hydrolysis by the enzymes of the human digestive system. Digestible carbohydrates are broken down into their monomers and absorbed from the small intestine, while non-digestible carbohydrates reach the colon. Another distinguishing feature is the use of carbohydrates reaching the colon by the colon microbiota. Carbohydrates that can be used by the microbiota can shape microbiota composition and function.

20In the cecum and right colon, fermentation is very intense with a high production of SCFAs (acetate, propionate, and butyrate), which generate an acid pH (range 5–6) and rapid bacterial growth. By contrast, the substrate in the left or distal colon is less available; the pH is almost neutral; the putrefactive processes become quantitatively more important, and the activity of the bacterial population is lower. Putrefaction is another anaerobic metabolism of peptides and proteins (elastin and collagen from food sources, pancreatic enzymes, exfoliated epithelial cells, and lysed bacteria, etc.); it also produces SCFAs but, at the same time, generates potentially toxic substances including ammonia, amines, phenols, thiols, and indoles.

21SCFAs (i.e. acetic, propionic, and butyric acids) are released because of the fermentation of some dietary components, such as dietary fiber by the colon microbiota. Current evidence shows that with low dietary fiber, microbial diversity is significantly reduced, and there are significant changes in microbiota composition. On the other hand, increasing dietary fiber intake increases intestinal microbial richness and/or diversity. It has also been reported to increase bacterial gene expressions for butyrate production and decrease genes required for secondary bile acid synthesis. It has been shown that long-term high fiber intake in the diet can shape the enterotype of the microbiota, especially prevotella colonization.

22SCFAs have important functions in the physiology of the host. To illustrate, butyrate is the main source of energy for the epithelium of the colon and appears to have a protective effect against colon cancer; acetate and propionate are metabolized by the liver (propionate) or by peripheral tissues, in particular by muscles (acetate), and can play a role as modulators of glucose and cholesterol metabolism; butyrate and propionate favor the aboral transition of feces. SCFAs have broad effects on many cell types, including myeloid cells with anti-inflammatory effects and regulatory T cells in the colon.

23SCFAs can exert powerful immunomodulatory effects by suppressing the activation of the nuclear factor-kB and/or by acting on the G protein-coupled receptors as demonstrated by the acetate and increasing the expression of the Treg gene. This effect translates into some studies of an increase in immunological tolerance and protection from inflammatory or allergic diseases. On the other hand, there is also very limited evidence that this fermentation may negatively impact colon cancer.

24Unlike the other cells of the human body, which use glucose as primary energy, the colonocytes, rapidly proliferating cells, exploit butyrate for 60%–70% of their energy. As a fatty acid, butyrate is beta-oxidized in mitochondria. Interestingly, butyrate has been shown to have a higher antimutagenic effect compared to organic acids (acetic acid, lactic acid, and pyruvic acid) produced by Lactobacillus and Bifidobacteria.

25According to Warburg’s hypothesis, cancer cells consume glucose, so the butyrate in these cells is not metabolized, accumulates in the nucleus where it inhibits histone deacetylases (HDAC), and therefore the cell cycle progression. Moreover, the bacteria that produce SCFAs seem to affect the enterocyte cycle in the colon; in particular, butyrate inhibits cell proliferation, stimulates differentiation in epithelial neoplastic cell lines in vitro, and promotes the return from neoplastic to non-neoplastic phenotype. The mechanisms underlying the cancer prevention of fiber are of course not related to butyrate alone. Insoluble fiber types such as cellulose are not fermented by intestinal bacteria and accelerate intestinal transit. Decreased intestinal transit rate is thought to be effective in preventing cancer in that they reduce exposure of colon cells and exogenous carcinogens (such as heterocyclic amines from meats).

26Finally, the colon microorganisms assisted by the presence of SCFAs also play a role in the synthesis of some vitamins (B1, B2, B3, B6, B12, pantothenic, biotin, and folic acid) and the absorption of some minerals such as calcium, magnesium, and iron.

27Esophageal cancers are the sixth leading cause of death. Normally, the number of microorganisms in the esophagus is low. It is reported that Streptococcus viridans is the most common in the esophageal microbiota. The transition from Gram (+) bacteria to Gram (−) bacteria predominance in the esophageal flora induces an important inflammatory response that triggers dysbiosis. Loss of bacterial diversity and atrophic gastritis are increasingly associated with distal esophageal cancer. Higher levels of Campylobacter concisus and Campylobacter rectus have been found in patients with premalignant Barrett’s esophagus and are associated with pathogenesis. In the scans performed on adenocarcinomas, it has been shown that the microbiota includes Campylobacter and some Escherichia coli species.

28The involvement of Helicobacter pylori in gastric cancer is the best-known example of a microbial infection related to cancer in the human gastrointestinal tract. In the early 1990s, these flagellated Gram (−) bacteria were classified in group I of carcinogens by the International Agency for Cancer Research.

29Several studies have shown that Helicobacter pylori are implicated in the onset of gastric adenocarcinoma; specifically, the deficiency of the aforementioned bacterium coincides with the increase in the incidence of this tumor form due to the consequent alterations in the physiology of the gastrointestinal tract (secretion of gastric acid, hormones, and immunocytes) and changes the composition of the microbiota since it alters the proportion of other microbial species present. Studies have shown that changes in microbiota diversity occur during the sequence of chronic gastritis-atrophic gastritis-intestinal metaplasia-dysplasia, with an increase of 21 groups, including Peptostreptococcus stomatis, Parvimonas micra, Fusobacterium nucleatum, and the depletion of 10 bacterial groups.

30Helicobacter pylori play a role in oncogenesis at the gastric level through three mechanisms. First, helicobacter pylori inject two cytotoxins, VacA and CagA into the host cell, which activates oncogenic signal transduction pathways. Secondly, it induces the production of reactive oxygen species (ROS) that activate inflammatory pathways. Finally, causing atrophic gastritis is characterized by the destruction of the parietal cells that produce acid so that there is a compensatory upregulation of gastrin that stimulates the cells to produce more acid, but also activates oncogenic signals. It is shown that Helicobacter pylori are responsible for chronic atrophic gastritis while it is the consequent change in the microbial flora responsible for the progression to gastric cancer. Following the reduction of gastric acidity, the carcinogenic power of some bacterial strains may increase. In addition to these, the microbiota is also populated with bacteria that reduce nitrates, it follows the formation of nitrites and carcinogenic N-nitroso compounds.

31Colorectal cancer is one of the most common cancers in the world, and it is a partly hereditary tumor, partly influenced by external factors and lifestyle. The colon has the most populous bacterial community in the human body with a prevalence of Firmicutes and Bacteroidetes and many more immune cells than other mucous membranes and lymphoid tissues. Moreover, the association between inflammation and cancer is particularly strong in colorectal cancer and indeed patients with chronic intestinal inflammation increase the risk of cancer by 2–10 times. Recent evidence shows that even some bacteria, such as Bacteroides fragilis, Escherichia coli, and Peptostreptococcus anaerobius could promote colorectal cancer by the activation of Th17 cells, direct DNA damage, and induction of cholesterol synthesis.

32It has not yet been defined what triggers the start of the mutations responsible for the transformation of adenocarcinoma, but from metagenomic studies on fecal microbiota and mucosal of the patient with colorectal cancer, a dysbiosis and an abundance of Fusobacterium, especially Fusobacterium nucleatum, Peptostreptococcus, Parvimonas, and Porphyromonas which are the components of the oral microbiota have been noted. This suggests that carcinogenesis is associated with bacterial translocation from the mouth to the intestine.

33Fusobacterium, in Fusobacterium nucleatum, is a commensal of the oral cavity, but if present in abundance in the colon-rectum, it is associated with metastasis of regional lymph nodes and tumor localization in the colorectal colon. Fusobacterium nucleatum promotes colon cancer through multiple mechanisms. Fusobacterium nucleatum has a direct interaction with epithelial cells; it attacks and invades epithelial cells with FadA adhesion molecules that bind E-cadherin on the cell surface and activates Wnt. It is internalized and activates other inflammatory genes. This mechanism is confirmed by the presence of high expression of the FadA gene in adenomas and colorectal tumors. With the Fap2 protein, Fusobacterium nucleatum also binds a receptor on T lymphocytes and natural killer (NK) cells and blocks the cytotoxic activity on tumor cells. Moreover, the bacterium could also modify the tumor microenvironment and induce the expression of microRNA-21. It is interesting to point out that the increase in Fusobacterium nucleatum is linked to different clinical and histological parameters, such as lower T cell infiltration, advanced disease status, and low survival. However, the difficulty is to understand the causal link: is the bacterium the cause of carcinoma or is it only the end-user of an environment altered by the tumor? There is no study confirming the cause and evidence that a transplant of specific bacteria can cause a tumor in the recipient as seen in obesity. However, there is quite convincing evidence of how extensive alterations of the bacterial flora led to the creation of a pro-oncogenic environment that favors the neoplastic transformation of the cells. This is the so-called hypothesis of the alpha bug, that is a small number of bacteria, acting as alpha subjects, would be the promoters of the carcinogenesis damaging the DNA and would favor the transformation of the remaining bacterial population of the organ concerned.

34The microbiota can also act at a distance because it can alter the intestinal barrier. The intestinal epithelium consists of a single layer of cells that separates the microbiota in the lumen from the intraepithelial lymphocytes and the cells of the innate and adaptive immune system present in the lamina propria. A thin layer of mucus produced by the goblet cells covers the epithelium of the colon and prevents direct contact of the microbes with the epithelium. A diet low in fiber can cause a condition of dysbiosis in which the butyrate-producing bacteria decrease and increase Akkermansiamuciniphilia and Bacteroides caccae which degrade the mucus. All this leads to a condition of a “leaky gut”, and therefore increased permeability of the intestinal barrier. Clinical and experimental data suggest the importance of intestinal hyperpermeability in the inflammatory changes of various diseases including GI cancers.

35On the other hand, several metagenomic studies have identified significant enrichment of butyrate-producing bacteria in healthy controls compared to those affected by colorectal cancer and in patients suffering from a reduction of commensal bacteria (Bifidobacteria, lactobacillus, Ruminococcus) and producing butyrate (Lachnospiraceae and Faecalibacterium). SCFAs enhance barrier functions and exert anti-inflammatory and tolerogenic effects on immune cells through mechanisms such as G-protein-coupled receptor (Gpr)-mediated sensitization of IEC inflammations and reduction of IEC oxygen concentrations and induction of hypoxia-induced factor (HIF). In this way, SCFAs have potent anti-inflammatory and anticancer effects. Butyrate suppresses carcinogenesis through multiple mechanisms; as a histone deacetylase inhibitor, regulates cell proliferation and apoptosis, binds G proteins implicated in tumor suppression and induces Treg cells, maintains the barrier function epithelial increasing the expression of tight junctions which are important for preventing inflammation. It has been found that Africans have an abundance of butyrate-producing bacteria and a reduced risk of colorectal cancer when compared to African Americans with a high risk. Faecalibacterium prausnitzii is the major producer of butyrate together with Clostridium IV and XIVa. Lachnospiraceae, another butyrate producer, is reduced in the feces of colorectal cancer patients. One could imagine that the reduction of butyrate-producing bacteria and other short-chain fatty acids may contribute to the progression of colorectal cancer. It is interesting to note that Fusobacterium also produces butyrate but uses it as a substrate, amino acids, for example, lysine, which develop ammonia which is harmful to the intestine.

36Except for all this information, the use of the microbiome in diagnosis is another question that needs to be answered. Given the spread of rectal colon cancer, biomarkers are being researched to allow the screening. Case-control cohort studies conducted in America, Europe, and China give hope that markers can be identified in the fecal microbiota as 20 genes closely associated with colorectal cancer have been found. One of these genes is that linked to the Fusobacterium nucleatum which also gives information on the prognosis, unfortunately negative. The fecal microbiome does not correspond perfectly to the mucosal one and is more useful for identifying risk factors associated with colorectal cancer. From the diagnostic point of view, the combination of 4 markers to beat us (Fusobacterium nucleatum, Clostridium Hathaway, Bacteriodes clarus, and an indefinite species “m7”) turns out to be more accurate than Fusobacterium nucleatum alone. Besides, comparative studies between healthy people and patients on the oral microbiota have also identified differences that could be used as markers to diagnose esophagus cancer and pancreas.

37These data, which are likely to be used in the clinic in the future, need to be investigated with further studies. Fecal microbiota analysis showed that there is an association between colorectal cancer and increased lipopolysaccharide metabolism (LPS) which increases the activation of inflammatory pathways by TLRs whose substrates are derived from various bacterial products (LPS, PAMPs).

38Possible mechanisms regarding the role of the gut microbiome in cancer development or prevention are schematized in Figure 1. There are many hypotheses about these mechanisms, and there are many factors that modulate this positive or negative effect. The relationship between cancer and microbiota has intrigued the biomedical community since the late 19th century when William Coley began treating sarcomas by injecting bacteria, the so-called “Coley’s toxin”. Subsequently, other microbial agents and their products were isolated to try to cure malignant tumors, such as bladder cancer via Mycobacterium bovis or melanoma via Herpes virus, or pancreatic cancer with Listeria monocytogenes. However, little has yet been proven regarding the role of microbiota in the development of gastrointestinal cancers. The current evidence is carcinogenesis of certain specific bacteria found in the gut shows that it can stimulate the immune system by showing modulating effects, stimulating inflammation, and increasing cell proliferation.

39Gut microbiome could modulate many systemic immune response elements such as the regulation of hematopoiesis, development of intestinal macrophages, and T cell response. The immune system participates in the initial phase of the suppression of malignant transformation, destroying the cells already infected with oncogenic viruses and recognizing the cells that express the tumor-associated antigens (TAAs). The immunogenicity of a tumor cell, and therefore its susceptibility to immunosurveillance, requires the combination of antigens, such as TAAs, and an immune system capable of responding to specific molecular patterns (DAMPs). The intestinal mucosa signals a threat to the innate immune system, through toll-like receptors that recognize and bind specific microbial macromolecules, and this triggers a protective response by commensal bacteria, an inflammatory response to pathogenic organisms, or the trigger of apoptosis. Therefore, the commensal bacteria of the gastrointestinal tract play an active role in the development and homeostasis of the immune system.

40The change in the diversity and distribution of bacteria is the most important factor that causes chronic inflammation and the onset of cancer progression. In the specific case of IBD, the mucus layer is thinner and less continuous, the tight junctions are destroyed, and the space between epithelial cells is increased. Therefore, the translocation capacity of the bacteria increases, and all this leads to the triggering of the recurrent chronic inflammatory process with the release of pro-inflammatory cytokines, Tumor Necrosis Factor (TNF), and Interferon-gamma (IFN-γ). Patients with IBD are more susceptible than others to develop colorectal cancer since chronic persistent inflammation determines an abnormal proliferation of tissue that first becomes metaplastic and then neoplastic. A few direct pieces of evidence in favor of the ability of commensal bacteria to promote tumor immunosurveillance are provided by the correlation of the growth and infiltration of major melanomas in mice with less expression in the microbiota of Bifidobacterium species.

41The increase in some tumors seems to be related to the reduction of certain microbial species, caused by modern lifestyles, which introduce ultra-processed foods and dysbiosis by frequent antibiotic therapies. Consistent with this, an epidemiological study conducted on 125441 people demonstrated the correlation between repeated use of penicillin, cephalosporins, or macrolides with lung, prostate, and bladder cancer, and tetracycline with breast cancer. Antibiotics likely favor the development of tumors by modifying the composition of the microbiota. Furthermore, the use of antibiotics during cancer therapies reduces their effectiveness. The mucosal barrier in this case has a double offense, on the part of chemotherapy and antibiotics, which can lead to the proliferation of opportunistic pathogenic bacteria that can move through the compromised epithelium and cause infection. There is also evidence on patients confirming that treatment with Gram (+) active antibiotics is associated with shorter progression-free survival and overall survival in patients with chronic lymphatic leukemia treated with cyclophosphamide and cisplatin.

42Identifying microbial agents that have a causal link with the process of carcinogenesis is an important issue since it was estimated that 15.0% of new cancer cases were due to infectious agents. The oncogenic mechanisms of these complex bacterial communities have not yet been fully clarified. However, there is also some evidence that some bacteria can produce toxins and metabolites that are involved in oncogenesis. Examples are CagA and VacA, the toxins produced by Helicobacter pylori, or deoxycholic acid, a metabolite of the genus Clostridium that predisposes to cancer of the colon and liver. In addition to some bacteria, there are also oncogenic viruses, such as the Papillomavirus, which are positively correlated with cancer of the cervix, head-neck, bladder, and uterus. Screenings of oncogenic bacteria and viruses in GI cancers may open areas of research for composing cancer control strategies.

43Nutrition can prevent or increase GI cancers by modulating the microbiota. Considering the positive restoration of gut modulation, the first thing that comes to mind is undoubtedly the MedDiet. The MedDiet is among the most healthy and sustainable diet models, which is a diet model mainly based on vegetables, fruits, legumes, nuts, beans, cereals, grains, fish, and unsaturated fats such as olive oil. It has been reported that compliance with the MedDiet model positively affects the gut microbiome ecosystem and has positive roles in ensuring the immune system and metabolic balance of the human organism. The MedDiet is based on regular consumption of monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs), polyphenols, and other antioxidants, high intake of prebiotic fiber and low-glycemic carbohydrates, and consumption of more plant proteins than animal proteins. Garcia-Mantrana et al. (2018) noted that a higher Firmicutes-Bacteroidetes ratio was associated with lower adherence to the MedDiet, whereas the greater presence of Bacteroidetes was associated with lower animal protein intake. Also, higher Bifidobacteria counts and higher total SCFAs were associated with greater consumption of plant-based nutrients such as plant proteins and polysaccharides. Consistent with all these, it has been reported in the literature that as adherence to the MedDiet increases, mortality is lower and the frequency of gastrointestinal system diseases such as NCDs, low-grade inflammation, and Crohn’s disease decrease. Studies investigating the effects of MedDiet on GI cancers, especially colon cancer, are intense. In this context, in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort, it was found that adherence to the MedDiet delayed the onset of colorectal cancer. Although the mechanisms associated with this are not clear, it has been reported that DNA methylation of cg20674490-RUNX3 may play a mediator role in this relationship. In another study, no negative relationship was found between MedDiet compliance and the incidence of colon, proximal colon, distal colon, and rectum cancers in the Dutch population, and this study shed light on the fact that many genomic and environmental factors can modulate this relationship. Contrary to MedDiet, the negative effects of Western diets on the gut microbiome ecosystem have been known for a long time. The low fiber but high fat and carbohydrate content of the Western diet is the basis of dysbiosis and related GI problems. Russell et al. administered a high-protein diet for 4 weeks, which resulted in a decrease in the total number of bacteria and microorganisms that produce butyrate, which is known as a protective agent associated with lowering cancer risk, such as Roseburia/Eubacterium rectale.

44Besides, the bacterial products that come from protein metabolism are thought to promote cancer and include N-nitroso compounds, ammonia, hydrogen sulfide, and polyamines. Roasted meat is of particular concern due to the formation of heterocyclic amines (HCAs) that are metabolized by colon bacteria in compounds that damage DNA. High-fat diets significantly reduced fecal SCFAs concentration compared to low-fat diets; it is also well known to stimulate secondary bile acid secretion and increase the fecal concentration of secondary bile acids such as deoxycholic acid (DCA). Changes in the microbiota with the increase in secondary bile acids increase both intestinal permeability and endotoxin production. This situation plays an important role in the physiopathology of many chronic diseases, including cancer. In addition, in the Western diet model, which includes frequently consumed processed foods, the intestinal barrier function may be further impaired by certain food additives, including dietary emulsifiers, and may cause significant health problems.

45There are also some foods and food components that are converted into onco-suppressive metabolites in GI. In the first place are the fibers and/or polysaccharides such as starch, cellulose, pectins, and gums, which reduce the risk of colorectal cancer through some mechanisms. The first mechanism is that insoluble fibers in food such as whole grains, green leafy vegetables, and dried fruits may assist in accelerating intestinal transit, therefore, reducing exposure to carcinogens that may be present. The second mechanism is that soluble fiber, contained in legumes, fresh fruit, and potatoes, sees the action of the microbiota in forming butyrate. Therefore, only soluble fibers generate butyrate, and only a long time dietary intake can bring important changes in the species of the microbiota that produce SCFAs. In addition to fibers, soy-based products such as tofu, miso, and soy milk, which contain daidzein (an isoflavone) in a form require a certain metabolism by the flora to be activated as an antioxidant. Interestingly, only 30%–40% of Westerners can metabolize daidzein in equol, while that percentage doubles in Asians. Cruciferous vegetables are another example of bioactive compounds such as glucosinolates and their hydrolysis products including indoles and isothiocyanates (ITCs), and accumulated literature has reported that high intake of cruciferous vegetables has been associated with a lower risk of certain types of cancer including colorectal cancer. When these raw vegetables are cut, the glucosinolates are converted by the myrosinase (thioglucosidase) into sulforaphane (SFN) which acts as an anti-inflammatory, as an HDAC inhibitor activity. However, the cruciferous are generally cooked and the heat denatures the myrosinases since that is temperature sensitive and can be inactivated upon exposure to temperatures over 60°C. However, human gut microbiota can secrete their myrosinase and can be a mediator to transform glucosinolate precursors in cruciferous vegetables to active isothiocyanates and in the colon, the bacterial thioglucosidases convert the glucosinolates into isothiocyanates which exert their beneficial effects. Finally, berries and berry constituents containing ellagic acid are metabolized to urolithin by microflora with an inhibitory function of ROS; cyclooxygenase-2 (COX-2), c-Jun (a component of activator protein-1 (AP-1)), inducible nitric oxide synthase (iNOS) expression; NF-κB and TNFα in preneoplastic tissues especially in the esophagus.

46The first strategies that come to mind in the prevention and treatment of GI cancers by modulation of gut microbiota or correction of dysbiosis are also biotics. Probiotics are identified as “live microorganisms that when administered in adequate amounts, confer a health benefit on the host”. The microorganisms most used as probiotics are the species from the genera Lactobacillus, Bifidobacterium, and other genera Bacillus, Propionibacterium, Streptococcus, Escherichia, and also Saccharomyces are also employed. They can be found and added to numerous types of products including foods and dietary supplements. Probiotic strains must be i) sufficiently characterized; ii) safe; iii) supported by at least one positive human clinical trial with accepted scientific standards and iv) alive in the product at an acceptable dose throughout shelf life. In the literature, it was shown that the involvement of probiotics in enhancing intestinal barrier functions, maintaining the integrity of narrow junctions, maturation of Tregs, activation of NK cells with direct elimination of bacteria, increasing mucin production from Globet cells, defensins from Paneth cells, secretion of IgA and anti-inflammatory cytokines from immune cells.

47Prebiotics are recently defined as “the substrates that are selectively utilized by host microorganisms conferring health benefits”. Plenty of fermentable carbohydrates have been suggested to include a prebiotic effect, but the dietary prebiotics frequently reported to promote health especially intestinal benefits are the fructans (fructooligosaccharides and inulin) and galactans (galactooligosaccharides) as non-digestible oligosaccharides. However, by the current definition, the term prebiotic not only covers oligosaccharides, but also includes other food components and metabolites (such as CLA, PUFAs, SCFAs, and dietary polyphenols), making them candidate prebiotics, additionally considering the benefits to regions outside the GI and includes various categories other than food. Ingestion of prebiotics is associated with anticarcinogenic effects. Prebiotics reduce pH via forming SCFAs-organic acids in the colon as they are fermented and/or degraded by enzymes (β-fructanosidase β-galactosidase), which are prevalent in Bifidobacteria, thus exerting anti-inflammatory activity, stimulating Treg cells, and reducing IFN-γ. Prebiotics are also able to inhibit the adhesion of pathogens to the intestinal epithelium, preventing translocation. Moreover, it has been shown that prebiotics increases the height of the villi, the depth of the crypts, the number of epithelial cells, and the thickness of the mucosal layer of the jejunum and the colon. Finally, another mechanism of anticancer activity is the detoxification of genotoxins such as nitrosamides and hydrogen peroxide in the gut. It plays a role in all these effects together with probiotics.

48In addition to probiotics, postbiotics have recently come to the forefront due to their lower risk compared to probiotics and their modulating effect on the microbiome. The International Scientific Association of Probiotics and Prebiotics (ISAPP) defined a postbiotic as a “preparation of inanimate microorganisms and/or their components that confers a health benefit on the host”. ISAPP has noted that effective postbiotics must contain inactivated microbial cells (micro and macromolecules) or cell fractions, with or without metabolites, that contribute to observed health benefits when administered in adequate amounts. In addition to its health benefits, there is evidence in the literature that it supports the treatment of colorectal cancer and reduces side effects by supporting and modulating immune system function. It is reported that it does this by showing antioxidant, antiproliferative, and anti-inflammatory effects. However, as it is still a new emerging topic, there is a need for longer-term, large-scale randomized-controlled studies investigating the effects of postbiotics on GI cancers.

49It is clear in recent years that the long-term response of anticancer drugs depends on the immune response, and the microbiome can play an important role in this. Therefore, in addition to the mechanism of carcinogenesis, the intestinal microbiota plays a role in mediating the results of anticancer therapies, giving hope for a potential improvement in treatment and reduction of toxicity through the manipulation of the microbiota. In preclinical studies, it was found that the composition of the intestinal microbiota correlates with antitumor immunity and therapeutic efficacy. For example, Bifidobacterium breve improves the efficacy of anti-programmed death 1 (PD-1), The cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4) blockade depends on some Bacteroides species, and platinum-based chemotherapy requires an intact intestinal microbiota. Indeed, it has been shown that a massive presence of Bifidobacterium improves the efficacy of anti-PD-1 in reducing melanoma growth through dendritic cells that become more active in presenting melanoma antigen to T lymphocytes. In a study, microbial correlations were found in response to anti-PD-1 therapy in patients with metastatic melanoma. Patients who respond to therapy have an abundance of Faelibacterium while those who do not respond will have an abundance of other Bacteroidales species.

50The oxaliplatin and cyclophosphamide chemotherapeutics are both less effective in tumor reduction in immunodeficient mice, in germ-free mice, or treated with broad-spectrum antibiotics. In a recent study of patients with non-small cell renal and urothelial lung cancer, the authors found a negative correlation between antibiotic use and response to anti-PD-1 therapy. These results corroborate the importance of intestinal microbiota in the response to immunotherapy and that microbial destruction by antibiotics may hinder the effectiveness of immune checkpoint blockade.

51The role of intestinal microbiota has also been investigated in chemoresistance, and it has been found that in the presence of Fusobacterium nucleatum autophagy mechanisms that confer resistance are activated. Furthermore, intestinal microbiota can predict whether a patient receiving ipilimumab will develop autoimmune colitis. For instance, the toxicity of immunotherapies can be mitigated by Bifidobacterium.

52In the last decade, the function of non-coding RNAs (ncRNAs) in cancer development has been clarified. They have a pivotal role in gene expression, cancer progression, and cell-cell communication through the involvement of extracellular vesicles (EV). ncRNAs have been classified into diverse classes based on their length, structure, and location. The important ncRNA types with well-defined roles in cancers are microRNAs (miRNA), circular RNAs (circRNA), PIWI-interacting RNAs (piRNA), and long-ncRNAs (lncRNA). Small RNAs known as miRNAs typically have a length of 20–22 nucleotides (nt). Targeted mRNA is degraded by the RNA-induced silencing complex (RISC) when miRNAs bind to the complementary region in the target mRNA. piRNA, which is 24–30 nt in length, was first discovered in the Drosophila. It is mostly found in germline cells and interacts with members of the PIWI family of proteins to help regulate chromatin’s epigenetic state.. LncRNAs and circRNAs are both longer than 200 nt, although circRNAs are ring-like whereas lncRNAs are linear. LncRNAs and circRNAs both fold into complicated second structures that allow them to interact with DNA, RNA, and proteins. They can both be created from exon, intron, intergenic region, or 5/3-untranslated sequences. ncRNAs can act as tumor suppressors and oncogenes in cancers and they are considered promising diagnostic and therapeutic markers. As of late numerous studies have appeared that the microbiota can influence the event and improvement of cancer by influencing the expression of ncRNA (Table 2).

53miRNAs play a crucial role in the relationship between the host and the microbiota in cancer. Numerous studies suggest that Helicobacter pylori infection may influence the host miRNA expression. Chang et al. showed that Helicobacter pylori-positive gastric cancer patients had considerably greater levels of miR99b-3p, miR-564, and miR-638 compared to Helicobacter pylori-negative patients, despite exhibiting significantly less of miR-204-5p, miR-338-5p, miR-375, and miR-548c-3p. MiR-18a-3p and miR-4286 levels were substantially larger in gastric cancer associated with Helicobacter pylori, according to research by Tsai et al. In a group of gastric cancer patients, serum expression of miR-18a-3p and miR-4286 was positively and significantly associated with Helicobacter pylori. Additionally, invasion, tumor stage, tumor size, and lymph node metastasis were all strongly linked with miR-18a-3p and miR-4286. Another study showed that Helicobacter pylori infection enhanced miR-223-3p expression, which in turn activated the NF-kB pathway and contributed to the development of gastric cancer. Interestingly, the Ebstein-Barr virus can affect cell signaling pathways, influence gene expression, cause methylation of the host genome, cause infected gastric epithelial cells to generate a tumor microenvironment, and initiate and progress gastric cancer. Ebstein-Barr virus-associated gastric carcinoma is a subtype of gastric cancer that has morphologic traits that are like those of cells going through the epithelial-to-mesenchymal transition. In one study, miR-200a and miR-200b, which are connected to the epithelial-mesenchymal transition and were assessed in gastric carcinoma, were expressed at lower levels in Ebstein-Barr virus-associated gastric carcinoma than in Ebstein-Barr virus-negative carcinoma.

54Numerous studies have demonstrated that the gut microbiome affects the expression of miRNAs and their target genes in colorectal cancer. According to one study, Fusobacterium nucleatum enhanced tumor cell miR-21 levels by turning on the TLR4-MyD88 signaling cascade, which in turn increased CRC cell growth and tumor development in mice. Other research has demonstrated that patients with high miR-21 and Fusobacterium nucleatum DNA levels consistently had a higher risk of negative outcomes. It has also been noted that Fusobacterium nucleatum enhances chemoresistance to CRC via changing autophagy in a way that is miRNA-dependent.

55Like Fusobacterium nucleatum, it has been shown that different Escherichia coli bacteria contribute favorably to CRC carcinogenesis in a way that is reliant on miRNA. Tan et al. demonstrated that probiotics could inhibit the expression of the oncogenes hsa-mir-153 and hsa-mir-429 and increase the expression of the tumor suppressors hsa-mir-140 and hsa-mir-132, whereas pathobionts could increase the expression of the oncogenes hsa-mir-223, hsa-mir-96, and hsa-mir-106a. The research revealed that the development of colorectal cancer and microbiota-mediated colorectal carcinogenesis depend on these miRNAs (Figure 2A, B).

56LncRNA is a subclass of non-coding RNA that has been linked to intestinal microbiota, tumor progression, and colorectal carcinogenesis. Four lncRNAs, including LINC00355, KCNQ1OT1, LINC00491, and HOTAIR, were found in the probiotic and pathobiont environments while two lncRNAs, including LINC00355 and KCNQ1OT1, were found in the pathogenic environment, according to Tan et al. Patients with CRC who express these lncRNAs strongly have poor prognostic value.

57Diet and dietary habits influence the microbiome, which is important in triggering the association between cancer and diet. On the other hand, tumor-associated-miRNAs can regulate the growth and composition of the gut microbiota along with the change in the metabolism. Metabolites produced by the microbiome can change gene expression including miRNAs (Figure 2C).

58The miRNA expression is regulated by gut microbiota, and aberrations in their expression can lead to pathological processes involved in cancer development and progression. MiR-17-92, miR-21, and miR-503 result to be more expressed in tumoral tissues compared with normal. MiR-21 reduces the expression of RAS p21 and programmed cell death-4. In CRC, infection of Fusobacterium nucleatum increases the expression of miR-21 and down-modulation of miR-18a and miR-4802. Crosstalk between microRNA-microbiota in the gut plays a pivotal role in gut homeostasis. microRNAs secreted by intestinal epithelial cells or diet-derived can influence the microbiota composition. There is a bi-directional regulation of miRNAs released and microbiota composition. Delivery of miRNAs may be explored as a therapeutic strategy due to the potential therapeutic interaction between miRNAs and bacteria (Figure 2D). Despite many studies, the gut microbiome and ncRNAs’ relationship and pathway mechanisms are still not fully known. Clarifying this process is very important for understanding the biology of cancer and developing treatment options.

59It has been shown that changes in the microbiota that cause dysbiosis are associated with various diseases, especially cancer. In recent years, it has been suggested that the gut microbiota, especially dysbiosis, or some specific microorganism species play an important role in the development of cancer, especially GI cancers. In this context, it is not easy to come to a definitive general judgment. Because many factors modulate this relationship, both host and microorganism, as well as the environment.

60The research has made important progress in the study of microbiota to facilitate the diagnosis and treatment of human diseases and to prevent them. Targeted microbiota interventions with natural foods and nutraceuticals including biotics may be used to prevent cancer, particularly in patients at risk and specific manipulations of the microbiota could be introduced into the clinic as an adjuvant regimen to increase efficacy and ideally to reduce the side effects of anti-cancer treatments. Of course, the most important issue in this application is to do this in expert monitoring and to consider the possible profit/loss ratio.

61The accumulation of scientific evidence offers opportunities for the development of new applications in cancer diagnosis and management. The microorganisms involved in the pathogenesis can offer valid biomarkers to get more information about the disease, the response to treatment, and its handling. Recent studies report that ncRNAs play an important role in cancer development by regulating several mechanisms involved in cell proliferation, differentiation, and apoptosis, including gut microbiome composition. ncRNAs represent promising approaches as diagnostics and therapeutics to explore in the future. However, further investigations are needed to deeply understand the role of ncRNAs in GI cancer.

62While waiting for further evidence to introduce the microbiota among the tools of precision medicine and nutrition, the most immediate approach is to dietary intervention enriching with fibers, and foods rich in bioactive substances and through pro-prebiotic foods, etc., limiting the consumption of processed foods and some certain type of animal foods. Along the same line, there is important evidence that the MedDiet is has a protective effect on GI cancers by modulating the microbiota. In addition to healthy nutrition practices, rational drug use strategies should be developed, and especially antibiotic treatments should be applied rationally to prevent all health problems related to dysbiosis, including GI cancers. Considering all these issues, it will be more realistic to develop microbiota-oriented strategies, especially in the prevention and treatment of GI cancers.