Introduction
Over the years, researchers tried to explain cancer pathogenesis by taking into consideration
different factors, such as immune system, the genetic predisposition and environmental
factors [1]. In this context, the inflammation and oxidative stress response represent
important topics in scientific debate. The inflammation is a normal defensal tissue
response to factors that disturb omeostasis of human biological system [1]. Therefore,
it needs a delicate regulation that ensures the correct reaction against damage and
allows the organ and tissue reparation associated to the recovery of their functions.
However, the correct operation of the “repairing machine in our body” is due to a
functional balance that allows the immune system to activate itself after physical
and/or chemical stimuli and then by determining its shutting down which ensures the
return to the starting conditions, essential for biological function protection. Response
of genetic factors to environmental stressors can complicate inflammation chronicization,
as well as the possible instauration of a chronic inflammation for autoimmune pathologies,
that represent the primum movens for cancer onset, just like inflammatory bowel disease
(IBD) [2]. The role of primary order carried out by immune system was first hypothesized
by Rudolf Ludwig Virchow in 1863, after leucocytes presence demonstration in neoplastic
tissue. This concept was taken up by a large number of studies on the topic, demonstrating
a clear interconnection among inflammation, oxidative stress, cytokine production,
chemokines and tumoral growth, invasion and metastasis [3]. The correlation between
inflammation and cancer is now very clear given the numerous scientific evidence especially
in gastrointestinal tract cancer [4]. In this context, an inflammatory microenvironment
is an essential assumption for the development of most of tumors. Indeed, only a small
part of cancers is correlated to germline mutation, whereas the majority is the result
of a cooperation between environmental factors and genetic somatic mutations [5].
The increase of metabolic pathologies, such as obesity and diabetes in the last years,
has caused an increase of tumor incidence in this population. These diseases are associated
to a higher risk of developing cancer due to a direct cancerogenous effect, as well
as an immune dysregulation able to promote chronic inflammation and oxidative stress
through antioxidant system depletion [6].
Additionally, also advanced age and cell senescence are important factor as they are
able to generate per se a higher predisposition to tumoral onset even through a dysregulation
of inflammatory cascade. These factors also make the immune response less efficient
in fighting cancer, allowing it to grow and metastasize [7].
Moreover, growing scientific evidence has demonstrated a possible role of gastrointestinal
bacterial community in the regulation of several inflammatory/immunitary processes
involved in tumor initiation or progression. Gut microbiota represents the totality
of bacteria, virus and fungi that are in our gastroenteric tract. It is a complex
ecological system consisting of at least 500 different bacterial species. Its qualitative
and quantitative composition is deeply different depending on the considered gastroenteric
tract. In the stomach, a small number of bacteria have been found, mainly consisting
of lactobacilli, streptococci, staphylococci, enterobacteriaceae and yeasts. In the
subsequent gastrointestinal tracts, there is a quantitative increase from 0 to 105
colony-forming unit/g (CFU/g) in the duodenum to 108 CFU/g in the ileum and 1010 CFU/g
in the colon. In the colon more than 99% of the microorganisms are strictly anaerobic,
such as bifidobacteria, Bacteroides spp., Clostridium spp., Eubacterium spp., Fusobacterium
spp., and peptostreptococci [8]. It varies from one person to another and is modified
by age, diet, type of birth, breastfeeding, ileocecal valve efficiency, use of active
drugs in both heartburn and gastrointestinal mobility [9], [10], [11], [12].
In order to understand the connection between gut microbiota and cancer, we have to
bear in mind that some of the functions carried out by a eubiotic gut are resistance
to intestinal colonization by pathogen bacteria able to cause dysbiosis, induction
in IgA production and antimicrobial secretions, regulation of structural entirety
of tight junctions and, above all, regulation of innate and adaptive immunity [13],
[14], [15], [16].
However, despite the growing interest regarding this field, nowadays, a standardized
protocol to study the relationship between gut microbiota and cancer, especially in
the interpretation of study results, does not exist. The strongest scientific evidence
concerning this relationship exists in relation to gastrointestinal tumors.
In this review, we analyze, from a particular perspective, the different ways to induce
inflammation and oxidative stress in order to explain the mechanisms underlying the
development of several cancers. Moreover, because of the strong scientific evidence
in literature, we focused our attention to the analysis of the role that gut microbiota
plays in gastrointestinal cancer development/promotion.
The Role of Tumoral Microenvironment
The major environmental factors responsible for induction of chronic inflammatory
process and cancer genesis are represented by chronic infection (such as viral hepatitis
for hepatocellular carcinoma development, Helicobacter pylori infection to gastric
cancer and mucosa associated lymphoid tissue development, schistosoma and bacteroides
infection to bladder and colon cancer), toxic exposition (e.g., tobacco, chemical
products and asbestos) as well as metabolic pathologies able to alter regulatory balance
of inflammation [17], [18], [19]. An important concept underlies the interconnection
between inflammation and cancer: the lasting inflammation. It would not be the intensity
of the inflammatory response but the maintenance of a low-grade and chronic inflammation
that determines the neoplastic transformation of eukaryotic cells or the induction
of genotoxic damage [20]. However, it is important to underline that not all chronic
inflammations, although systemic, are able to promote carcinogenesis. The development
of solid tumors is associated to an intrinsic tumoral inflammation supported by protumorigenic
microenvironment [5]. The tumoral microenvironment is composed by macrophages, neutrophils,
mast cells, myeloid-derived suppressor cells, dendritic cells, and natural killer
cells; moreover, adaptive immune cells (T and B lymphocytes) in addition to the cancer
cells and their surrounding stroma (fibroblasts, endothelial cells, pericytes, and
mesenchymal cells). These cells control tumor growth communicating through the production
of autocrine, paracrine and endocrine mediators, thus controlling tumoral growth.
The different types of interconnection, the grade and the type of cellular activation
are factors that conditionate the interplay between immune system and the tumor in
terms of tumor-promoting or antitumor response [21], [22]. It is possible to hypothesize
that the two types of tissue inflammation can coexist in natural history of tumoral
pathology. The role that this inflammation would have in tumor would be dictated,
in one way or another, by a specific composition and activation of the tumoral microenvironment
[23].
Macrophages could have a controversial role in this field. In fact, in relation to
the acquisition of a specific functional phenotype, they could have a tumor-promoting
or antitumoral activity. They can range from proinflammatory, antitumorigenic M1-phenotype
to anti-inflammatory, protumorigenic M2-phenotype macrophages (fundamental for the
induction of tumoral angiogenesis and for invasion and metastatic processes) [24].
T lymphocytes could have a similar role. In fact, they are divided into several subtypes:
CD8+ cytotoxic T cells (CTLs) and CD4+ helper T (Th) cells, which include Th1, Th2,
Th17, and T regulatory (Treg) cells, as well as natural killer T (NKT) cells. An increase
of CTLs and Th1 in tumoral microenvironment seems to be related to better prognosis
as highlighted in several studies on colon and pancreatic cancer, and a decrease in
their amount in tumoral microenvironment is associated with a greater chance of spontaneous
cancer development or as a result of exposure to toxic environmental factors [25],
[26], [27].
However, controversial results of a possible procancer role played by T lymphocytes
(CTLs, Th1, Th2 and Th17), except NKTs, are present in the literature [28], [29],
[30]. Some oncogenes such as RAS and MYC would be able to trigger a signal transduction
cascade that leads to recruitment of additional leukocytes in the tumor region and
the expression of inflammatory cytokines and chemokines, as well as the production
of angiogenic factors [31], [32]. Often, the necrosis developed in the core region
of a fast-growing solid tumor induces the release of proinflammatory mediators such
as interleukin (IL)-1 and High Mobility Group Box 1 (HMGB1) [33], which in turn causes
the release of angiogenetic factors, thus promoting cancer survival either directly,
by the increase in oxygen supply and nutrients to tumor tissue, and indirectly, by
recruitment of proinflammatory cells and cytokines release [34]. Other tumors are
able to stimulate inflammation through the direct production of proinflammatory substances,
some of which activate macrophages through interaction with Toll-like receptor (TLR)-2
[35]. Another type of inflammation associated with the tumor is that provoked by antitumor
therapy. It can represent the result of tissue necrosis with the consequent release
of proinflammatory cytokines that increase tumor growth or, on the other hand, can
be the antitumor response elicited by an increased exposure of tumor antigens to an
immune system by radio and/or chemotherapy [36], [37].
Dysregulation of the inflammatory system is also involved in the tumor initiation
process. A mechanism associated with inflammation-induced tumorigenesis is the upregulation
of activation-induced cytidine deaminase (AID) that is an enzyme able to induce the
switching of immunoglobulin gene class [38]. The expression of this enzyme is related
to an innumerable series of tumors and is linked to the activation of inflammatory
signals associated with exposure to environmental factors such as NF-kB or TGF-β–dependent
pathways [38]. The overactivation and overexpression of AID lead to the induction
of genetic instability that is the basis of the acquisition of new DNA mutations responsible
for the tumor initiation process, especially if loaded with critical genes such as
Tp53, c-Myc and Bcl-6 [39]. In this scenario, epigenetic regulation of gene expression
is also crucial. Epigenetic mechanisms including micro-RNA–based silencing and DNA
methylation are influenced by the onset of chronic inflammation responsible for the
silencing of oncosuppressive genes such as INK4a and APC [40], and in particular,
aberrant CpG island methylation in tumors is related to an increase of cancer development.
An important factor able to induce tumor process and to acquire uncontrolled replication
capacity by stem clone cells is represented by inflammation-induced production of
growth factors and cytokines. As demonstrated by Oguma et al., TNF-α would indeed
induce penetration within the β-catenin nucleus in conjunction with the inflammation-associated
gastric cancer genesis in the absence of any type of mutation of Wnt/β-Catenin pathway
[41]. On the other hand, the same DNA damage would be able to induce inflammation,
which in turn would carry out procancerogenic activity. In a diethylnitrosamine-induced
hepatocarcinoma model (DEN), DNA damage has been shown to induce cell death and necrosis,
responsive to the release of DAMPs and thus stimulation of Toll-like receptors, resulting
in inflammation activation [42], [43]. The activity of different types of oncoproteins
such as Ras, Myc and RET may also trigger signal activation that can trigger the production
of proinflammatory cytokines and chemokines such as IL-6, IL-8, IL-1β, etc. [5]. From
this evidence, it is clear that complex relationship between inflammation and cancer
is the basis of the meccanism that allows tumorigenesis and sustains disease progression
up to the acquisition of metastatic capacity.
The Role of Chronic Inflammatory Diseases
Epidemiological studies have shown that chronic inflammation predisposes individuals
to various types of cancer. It is estimated that underlying infections and inflammatory
responses are linked to 15% to 20% of all deaths from cancer worldwide [44]. There
are many triggers of chronic inflammation that increase the risk of developing cancer.
Such triggers include microbial infections (for example, infection with Helicobacter
pylori is associated with gastric cancer and gastric mucosal lymphoma or HCV infection
which is associated with liver carcinoma), autoimmune diseases (for example, intestinal
bowel disease (IBD) is associated with colon cancer) and chronic inflammatory conditions
such as asthma, chronic obstructive pulmonary disease (COPD). Accordingly, treatment
with nonsteroidal anti-inflammatory agents decreases the incidence and the mortality
from several tumor types [45], [46], [47]. In particular, nonsteroidal anti-inflammatory
drug use decreased cancer incidence in healthy population [48], and celecoxib demonstrated
an effect on colorectal polyps in patients affected from familiar adenomatous polyposis
syndrome [49].
One of the best studied example is colon cancer on IBD, in which colon cells are continuously
exposed to growth-promoting inflammatory cytokines [50]. The increased risk of cancer
in patients with IBD may be associated with the chronic proliferation required to
repair damage to the epithelial monolayer caused by constant inflammation. In chronic
inflammation, cytokines secreted by immune cells stimulate pathways [51], [52], [53]
which are essential for cancer proliferation.
Tumor necrosis factor α (TNFα) plays a fundamental role in inflammation in IBD and
has been the target of biological treatments. TNFα provokes inflammation by stimulating
the production of IL-1β and IL-6, inducing expression of adhesion molecules, proliferation
of fibroblasts, activation of procoagulant factors, and cytotoxicity of the acute-phase
response [54]. The binding of TNFα to its receptor causes the activation of mitogen-activated
protein kinases (MAPKs) and of the NF-ĸB pathway, which may influence barrier permeability.
NF-ĸB activation leads to increased transcription of proinflammatory cytokines, resulting
in a continuous feeding of the inflammatory response, and increased expression of
myosin light chain kinase (MLCK) [55], [56], which in turn stimulates permeabilization
of the intestinal barrier. Additionally, TNFα seems to stimulate the COX2-derived
PGE2 which may have a direct impact on carcinogenesis through regulation of the WNT
signaling pathway [54].
Another cytokine involved in the evolution of cancer is TGFβ1 that plays an important
role in the epithelial-mesenchymal transition (EMT) [57], [58], thus inducing a migratory
and apoptosis-resistant phenotype of intestinal epithelial cells through SLUG induction
and subsequent L1CAM gene expression.
Of interest, several reports have revealed that, in addition to the cytokine proinflammatory
effects, also several molecular alterations are involved in the inflammation-induced
carcinogenesis which involve inactivation of tumor-suppressor genes, oncogene mutations,
loss of heterozygosity, and chromosomal and microsatellite instability (MSI).
The TP53 tumor-suppressor gene appears to be a key factor in the early stages of IBD-associated
colorectal carcinogenesis, as it develops early in patients with IBD, whereas it occurs
later in sporadic colorectal cancer (CRC) [58]. It seems that TP53 abnormalities are
driven by inflammation, this hypothesis being supported by the presence of increased
TP53 expression in inflamed, nondysplastic, noncancerous colonic mucosa in IBD [59].
A very early and gradually occurring event in cancer development is the genomic instability.
Indeed, in the subset of patients with IBD, the colonic epithelium is damaged by reactive
oxygen species (ROS) produced in the context of the inflammatory milieu as a consequence
of the oxidative stress, leading to cellular damage in terms of oxidation of proteins
and DNA. Failure to remove or repair ROS-initiated damage can be either mutagenic
or lethal to cells [60].
In absence of coding-region mutations, often epigenetic changes, represented by aberrant
promoter methylation, occurring in association with silencing of tumor-suppressor
genes [e.g. TP53, Kruppel-like factor 6 (KLF6), APC, KRAS, and deleted in colorectal
cancer (DCC)] have been detected in patients with IBD-associated cancer [61]. In the
context of the progression of inflammation in IBD, DNA methylation is a key factor
in a subset of tumors affected by the CpG island methylator phenotype, a pathway that
emerges as a form of epigenetic instability [62]. In this scenario, an important aspect
of IBD-associated neoplasms is the defective DNA mismatch repair, manifested as microsatellite
instability (MSI) and promoter hypermethylation of the mismatch repair gene mutL homolog
1 (MLH1) [63]. Recently, epigenetic abnormalities and aberrant methylation have been
demonstrated also to alter those signal pathways involved in the stem cell proliferation
and differentiation capacity, such as WNT, NOTCH and HEDGEHOG pathways. In particular,
methylation of the WNT-signaling genes has been shown in early-stage IBD and to gradually
rise during progression of IBD-associated CRC.
Another inflammatory condition associated with cancer development is the COPD which
represents an abnormal and chronic inflammatory response of the lungs to noxious particles
and gases. Of note, approximately 30% of patients with mild to moderate COPD have
been reported to die from lung cancer [64], which has traditionally been linked to
a common etiological exposure, namely, tobacco smoke. The chronic injurious state
of this lung microenvironment may facilitate tumor development progressing from metaplasia,
dysplasia, carcinoma in situ and subsequent malignant transformation [65]. Morphological
changes in the bronchial epithelium are accompanied by an increase in loss of heterozygosity
and field cancerization involving the accumulation of mutations that eventually predispose
the lung to cancer [66], [67], [68]. Similarly to the intestinal IBD-associated carcinogenesis,
TP53 mutations characterize the smoker epithelium exhibiting squamous metaplasia and
occur early during transformation [66]. Microsatellite instability (MSI) is frequent
in the nonmalignant bronchial epithelium of COPD and is associated with EGFR amplification
[69]. The increased EGFR expression [70] and EGFR transactivation, observed in COPD
epithelium, augment and prolong inflammatory responses initiated by viral and bacterial
infection in the bronchial epithelial cells [71], [72]. Likewise, dysregulation of
the PIK3CA/PTEN/Akt/mTOR signaling, which coordinates tumor-promoting survival, metabolism,
migration and angiogenesis, is also observed in the bronchial airway of smokers with
dysplastic lesions, suggesting an early activation during carcinogenesis [73]. Indeed,
amplification of PIK3CA has been detected in high frequency in squamous cell carcinoma
(SCC), in contrast to gain-of-function mutations that are much less frequent [74],
whereas loss of PTEN protein expression or PTEN promoter methylation has been evaluated
as an independent poor prognostic factor for patients with non–small cell lung cancer
(NSCLC) associated with a more aggressive subset of lung tumors [75], [76], [77].
These examples of inflammation inducing cancer can be separated from the tumor-elicited
inflammation, which occurs when the invasive tumor is already established and drives
invasion and metastatic processes. In fact, the connection between inflammation and
cancer can be viewed as bidirectional: an extrinsic pathway, driven by inflammatory
conditions that increase cancer risk (such as IBD and COPD); and an intrinsic pathway,
driven by genetic alterations that cause inflammation (such as oncogenes). In the
latter case, the genetic events that cause neoplasia are also responsible for generating
an inflammatory environment.
The most frequently mutated oncogenes in human cancer are represented by both MYC
and members of the RAS family, and in turn components of the RAS–RAF signaling pathway,
which are able to induce the production of tumor-promoting inflammatory chemokines
and cytokines [32], [78], [79] and additionally to remodel the extracellular microenvironment,
such as inducing angiogenesis [31], [80].
An example in which to explore the connection between oncogenes and inflammatory microenvironment
is human papillary thyroid carcinoma, where the rearrangement of the RET gene is a
frequent early event in the pathogenesis of carcinoma and is a necessary and sufficient
event for this cancer to develop. The activation of RET induces a transcriptional
program that is similar to that which occurs during inflammation and includes colony-stimulating
factors (CSFs), which promote the survival of leukocytes and their recruitment from
the blood to the tissues; interleukin 1β (IL-1β); cyclooxygenase 2 (COX2); chemokines
that can attract monocytes and dendritic cells (CC-chemokine ligand 2 (CCL2) and CCL20);
chemokines that promote angiogenesis (such as IL-8; also known as CXC-chemokine ligand
8 (CXCL8)); the chemokine receptor CXC-chemokine receptor 4 (CXCR4), which binds to
CXCL12; extracellular-matrix-degrading enzymes; and the adhesion molecule lymphocyte
selectin (L-selectin). These results show that an early genetic event that is necessary
and sufficient for the development of a human tumor cancer directly promotes the build-up
of an inflammatory microenvironment.
Which pathway, extrinsic or intrinsic, between inflammation and cancer would be more
significant may be dependent on tumor type, or maybe, both are both essential. This
is the case of pancreatic cancer in a murine model, where both pancreatitis and K-RAS
gene mutations are frequently found and both are required to induce pancreatic intraepithelial
neoplasia and invasive ductal carcinoma [78]. Thus, although the RAS-RAF pathway [32],
[79] can drive tumor-promoting inflammation, an extrinsic inflammatory condition (pancreatitis)
is needed to drive carcinogenesis in mice and presumably in humans.
It is likely that all tumor-promoting inflammation, whether it precedes or follows
tumor development, is part of the normal response to injury and infection that has
been usurped by cancer cells to their own advantage.
The Role of Oxidative Stress as a Bridge to Link Inflammation and Cancer
The main known mediator of the link between inflammation and cancer is the imbalance
of oxidative stress induced by inflammation in a normal tissue and sustained by microenviromental
inflammation in a context of malignant tumor.
Reactive oxygen species derivates (ROS or intermediates ROI) are produced from molecular
oxygen O2 that is normally unreactive but is reduced to water through step-by-step
reactions generating partially reduced and very reactive intermediates with oxidizing
potential: the superoxide radical (O2
−), hydrogen peroxide (H2O2) and the hydroxyl radical (OH) [81]. Similarly, reactive
nitrogen species (RNS or intermediate RNI) are derived from nitrogen metabolism: NO,
synthesized by the enzyme NOS (nitric oxide synthase), nitrogen dioxide (NO2) and
peroxynitrite (ONOO-), from interaction of NO and O2, activator of nitrosylation [82].
These free radicals react with other chemical or structural compounds of cells and
also recruit other inflammatory cells with secondary amplification of damage.
Physiologically, the main sources of reactive species in all cells are mitochondria,
cytochrome P450 and peroxisome. Under physiological conditions, there is a constant
endogenous production of reactive intermediates of ROI and RNI that interact as “signaling”
molecules for metabolism, cell cycle and intercellular transduction pathways. The
production of ROI and RNI is balanced by a removal performed by a series of protective
molecules and systems globally defined as “antioxidant defenses,” such as superoxide
dismutase, catalase, glutathione peroxidase and glutathione-S-transferase. When the
generation of free radicals and active intermediates exceeds the system's ability
to neutralize and eliminate them, the oxidative stress occurs. In these conditions,
ROI and RNI act as “toxic” substances that may react with proteins, carbohydrates
and lipids, with consequent alteration in both the intracellular and intercellular
homeostasis, leading to possible cell death and regeneration. In the context of chronic
inflammation, the start of ROI and RNI-mediated carcinogenesis may be direct (oxidation,
nitration, halogenation of nuclear DNA, RNA and lipids), or may be mediated by the
products of ROI-RNI and proteins, lipids and carbohydrates that are capable of forming
DNA adducts. Proteins are more susceptible to oxidation by free radicals, where the
oxidation of SH groups of cysteine reduces the activity of various enzymes as well
as the synthesis of GSH, which is the main intracellular free radical scavenger. The
oxidation of lipids induces the formation of aldehydes and lipid peroxides that in
high concentrations are considered the more damaging species as they easily react
with proteins, DNA and phospholipids, generating a variety of intra- and intermolecular
toxic covalent adducts leading to the propagation and amplification of oxidative stress.
ROI can also increase the expression of transcriptional factors including c-fos and
c-jun oncogenes involved in neoplastic transformation and are able to recruit other
inflammatory cells, thus intensifying the possibility of DNA mutations in normal tissue,
leading to cancer development and proliferation [83]. In particular, in this peculiar
stress condition, intracellular pathways NF-KB, AP-1, p53 and caspases are activated
in terms of proangiogenic and antiapoptotic signals [84], [85]. Collectively, these
events lead to a state of a more complex oxidative and metabolic stress, with implications
in the control of cell regeneration at various level of DNA gene expression: mitochondrial
DNA (mtDNA) is very sensitive to oxidative stress because it lacks of histone proteins
and contributes itself to amplify radicals production in mitochondrial electron transport
chain [86], [87]. A proof of the relevance of role of oxygen and nitrogen species
as endogenous proinflammatory and procarcinogenetic agents is the consistent number
of experimental evidences that alterations in components of anti- and pro-oxidative
cascades have an impact on cancer development [88]. Lung cancer represents itself
the best example of chemical-induced oxidative stress in a context of chronic inflammation
as COPD [89]. Continuous cigarette smoking exposes pulmonary cells to over 4700 different
chemical compounds including 1015 oxidants and free radicals [90], and 69 carcinogenic
like tobacco-specific nitrosamines, formaldehyde and benzene [91]. Elevated levels
of ROS are produced normally by inflammatory and endothelial cells in response to
tobacco and pathogens [92], concomitantly with inhibition of the transcription nuclear
factor erythroid 2-like 2 (NFE2L2 or NRF2) [93] that encodes for cytoprotective and
antioxidant genes [94]. ROS compounds interact with polyunsaturated fatty acids and
generate reactive carbonyl species, able to suppress cellular PTEN, with subsequent
constitutive activation of protumoral AKT signaling [95]. These data suggest that
various biological mechanisms are associated with cigarette smoking–induced cancer
[96] in different lung tumor histological subtypes, depending on modulation of DNA
oxidative status.
The Role of Gut Microbiota in Gastrointestinal Cancer
More than 90% of gut microbiota consists of two dominant phyla (Bacteroidetes and
Firmicutes). The remaining part consists of five subdominant phyla: Actinobacteria,
Proteobacteria, Fusobacteria, Cyanobacteria and Verrucomicrobia [97], [98], [99].
However, phyla evaluation in intestinal microbial composition is an approximation
that does not consider the functional role carried out by the single bacterial species.
For this reason, nowadays, the use of information deriving from outcome of scientific
trials is scarcely applicable in routine clinical practice. Another strong limitation
in the management of gut microbiota for the treatment of several pathologies is represented
by the difficulty in modifying selectively the proportion of its microbial species,
without modifying the balance able to maintain “the gut health” [100]. To maintain
gut microbiota health means to maintain a correct proportion, which is different from
one person to another, among different bacterial species. This allows defending metabolic,
structural and immune functions of eubiotic microbiota, thus becoming essential for
the host life [13], [14], [15], [16], [101], [102]. The communication between “gut
microbiota system” and liver, brain, adipose tissue and other organs of human body
is regulated by intestinal permeability (IP). Its increase, in relation to a dysbiotic
gut, can be considered a key point for the comprehension of some pathogenetic mechanisms
of systemic and liver disorders as well as cancer. In addition to food adsorbed by
intestinal surface, bacterial products such as bacterial DNA, peptidoglycans (molecules
belonging to the class of pathogen-associated molecular patterns-PAMPs) and, in some
cases, intact bacteria can reach the liver in elevated quantities in relation to IP
levels (Figure 1) [103]. The clear division between what is located in our intestine
and portal blood is guaranteed by mechanisms able to regulate IP. Over time, the scientific
findings in this field have led us to understood that IP is not a static phenomenon
and mucosa intestinal is not a simple physical wall placed between intestine and portal
blood. IP degree is very variable and is interconnected to several factors: type of
diet, gene expression, intestinal/liver pathology, production of surface mucus, integrity
of tight junctions, production of immunoglobulins (Ig)-A. Many of these factors are
dependent from gut microbiota, which is the “lead” in IP control [16].
Figure 1
Mechanism of bacteria-induced inflammation through inflammasome activation.
Bacterial products such as bacterial DNA and peptidoglycans (molecules belonging to
the class of pathogen-associated molecular patterns [PAMPs]) reach the liver in a
large amount in relation to the reduction of gut impermeability. They can activate
innate immunity system through toll-like receptor binding and determine the recruitment
of several transduction pathways such as Myeloid differentiation primary response
88 (MYD88) related pathways with the subsequent activation of Phosphatidylinositol-4,5-bisphosphate
3-kinase (PI3K), mitogen-activated protein kinase (MAPK), Interferon regulatory factor
3 (IRF3), Protein Kinase B (Akt) and Nuclear factor-kappa B (NF-kB). All these proteins
are associated all together in a system of signal transduction called inflammasome
that regulates the cellular reaction to several molecules identified through toll-like
receptors (TLR) on cellular surface. The result is the activation of several types
of cells in production of inflammatory cytokines: interleukin (IL)-1β, IL-18, IL-6,
IL-12, transforming growth factor beta (TGF-β), and tumor necrosis factor alpha (TNF-α).
A specific composition of gut microbiota is correlated with some types of gastrointestinal
tract tumors through the activation of inflammasome involved in cancer development.
Moreover, the scientific literature is also enriched of association studies that tried
to link a specific composition of gut microbiota, in terms of prevalence of bacterial
species, to oncologic tissues abnormalities.
Figure 1
As demonstrated by Cariello et al., there is a relationship between IP, portal hypertension,
alcohol use, plasma levels of proinflammatory cytokines, and nitric oxide, expressed
as nitrosothiols, and nitrite levels in patients with various types and degrees of
chronic liver diseases. The assessment of IP degree through lactulose/mannitol test
on 83 patients with chronic liver damage showed an increase directly proportional
to the severity of liver disease. Independent factors from IP alteration were age,
portal hypertension, alcohol use, and diabetes. Moreover, plasma levels of inflammatory
cytokines and nitrosothiols were significantly higher in patients with altered IP
[104]. Cancer represents the second cause of death worldwide, and particularly, gastrointestinal
cancer is the leading cause of morbidity and mortality in the United States [105].
In recent years, there has been an increase in scientific research to investigate
the role of microbiota and microbiota-linked inflammation in carcinogenesis [106],
[107].
A possible strategy to explain the link between microbiota and cancer could be the
“Molecular Pathological Epidemiology” which consists in a multidisciplinary study
approach regarding the relationship between exogenous and endogenous factors (e.g.,
genetics) in the onset and progression of the tumor. Thanks to this view, it is possible
to study the capability of a specific environmental factor to induce changes in the
genetic expression that could influence cancer onset and its progression or prognosis
[108], [109]. Certainly, this approach has allowed us to make a lot of progress regarding
the understanding of the molecular mechanisms underlying neoplastic diseases such
as colon cancer, allowing, moreover, to identify clusters of population with high
risk to develop cancer and therefore deserving of more efficient screening surveillance
[110]. A classic example of how the microbiota can promote or not the organization
of the tumor by interacting with the environment could be its ability to exert an
anti-inflammatory or proinflammatory power through the production of metabolic substances
from the metabolism of diet components, inducing or not the generation of a tissue
microenvironment favorable to cancer development [111]. Tumor progression is also
associated with gene environment interactions. Recent advances in high-throughput
technologies gave us the possibility to understand better the link between a specific
microbial composition, the inflammation and the genetic modifications able to induce
cancer [112].
The conduction of studies in this regard is enormously difficult because of the extreme
degree of variability in the intestinal microbial composition between the analyzed
individuals, and for this reason, there is, however, no univocal opinion to date,
and future studies are needed in order to better understand the mechanisms implemented
by specific microbial species in the induction of gastrointestinal cancer.
A large number of evidence have been produced in recent past years about the viability
of the gut microbiota to influence the outcome of the therapy against different types
of tumors. In this regard, the functional link between gut microbiota and immune system
has been confirmed by the direct role of bacteria in influencing the efficacy of PD-1-based
immunotherapy against epithelial tumors [113]. The alteration of the intestinal microbial
composition induced by the use of antibiotic has been associated with a lower efficacy
of the immunotherapeutic regimens against this category of tumors [113]. In particular,
the relative proportional deficiency of Akkermansia muciniphila was correlated with
a lower response to therapy [113]. Akkermansia muciniphila restored the efficacy of
PD-1 blockade in an interleukin-12dependent manner by increasing the recruitment of
CCR9 + CXCR3 + CD4+ T lymphocytes in tumoral microenvironment.
The proportion of Fusobacterium nucleatum in some recent studies has been found to
be higher in patients with colorectal cancer [114], [115]. Its abundance also correlates
with a higher rate of relapse after chemotherapy and would be associated with a worse
prognosis of the disease. The relative abundance of this bacteria has also been correlated
with a lower response to chemotherapy with 5-fluorouracil, capecitabine and oxaliplatin.
Such phenomena would be linked to the interaction that such beat would have in regulating
the response to activation of TLR-4–mediated pathway and MyD88 inducing cellular autophagy
with the subsequent reduction in the tumor microenvironment of immune cells able to
fight the tumor [116], [117].
In the latter years, there was an increase in the incidence of esophageal adenocarcinoma
(EA) and the gastric esophageal junction carcinoma that showed an inverse proportional
relationship with the incidence of HP infection. This correlation has suggested that
HP infection could play a protective role against EA due to the ability to induce
hypochlorhydria as a result of the destruction of gastric parietal cells and the generation
of atrophic gastritis [118], [119]. In itself, inflammation caused by esophageal gastrointestinal
reflux on the distal part of the esophagus mucosa and the resulting metaplasia in
the long run are able to altering the composition of the esophagus flora. In an interesting
research, the microbial composition of biopsy samples of the esophagus was analyzed
by bacterial 16S ribosomal RNA gene survey and showed two different types of microbiome
in relation to the esophagus histology. In particular, the type I microbiome was dominated
by the genus Streptococcus and was related to the phenotypically normal esophagus;
on the contrary, the type II microbiome, composed for the greater part from gram-negative
anaerobes/microaerophiles, was related to esophagitis and Barrett's esophagus [120].
However, further studies are necessary to understand the concrete role of EC induction
variation. Gastric cancer represents the third cancer-related mortality cause worldwide,
and its incidence has changed in the last years, becoming less frequent in the distal
portion of the stomach (antrum and gastric body) and more frequent in the proximal
region (esophagus-gastric junction) [121]. The most important and known risk factor
for the development of gastric cancer is represented by Helicobacter pylori (HP) infection
[122]. This evidence is confirmed by the higher rate of eradication associated to
new therapies for HP infection that determined a clear reduction of gastric cancer
(GC) incidence rate. Between 1% and 3% of patients with HP infection develop a gastric
adenocarcinoma [123]. Among the factors able to trigger GC onset in patients with
HP infection, an important role is carried out by HP-oncoprotein cytotoxin-associated
gene A (CagA) in determining induction to progenitor cell proliferation in the gastric
mucosa [123]. Other factors that correlated HP and CG directly are represented by
secreted vacuolating cytotoxin A (Vac A) [124]. In particular, strains containing
type s1, i1 or m1 alleles within the 5′ region of Vac A gene are mainly related to
GC onset [125]. Moreover, HP could carry out an indirect activity in cancerogenesis
process for CG. Gastric mucosa colonization by HP is able to cause a reduction of
gastric mucosal flora richness: HP DNA represents about 93% to 97% of genetic sequence
isolable from genomic analysis of mucosal gastric flora in HP-positive patients, and
a total of 33 phylotypes were detected. By contrast, in HP-negative patient biopsy
samples, it is possible to identify about 262 phylotypes [126]. This variation in
gastric mucosal composition could have a role both in hypochlorhydria induction, which
represents the key role for histological progression to intestinal-type GC, and in
generating inflammation due to different microbic metabolism of nutrients ingested
with diet that could have a role in cancer pathogenesis through inflammation [111],
[127]. Gastric microbial composition modifies significantly itself in relation to
mucosa damage from chronic gastritis to intestinal metaplasia and GC. Indeed, as the
chronic gastritis passes from the GC, there is a marked reduction in the diversity
of gastric mucosal flora: Bacilli and Streptococcaceae are mainly prevalent in GC
samples compared with chronic gastritis and intestinal metaplasia samples. By contrast,
Epsilonproteobacteria and Helicobacteraceae family are less represented in samples
[128]. Some researchers highlighted a deep difference in terms of gastric mucosal
composition among patients who live in high-risk areas and low-risk areas for GC development
in Colombia. Two operational taxonomic units (OTUs), Leptotrichia wadei and a Veillonella
sp., were more present among patients who live in high-risk geographic area for GC
development, while 16 OTUs, including a Staphylococcus sp., were significantly more
abundant in patients who live in low-risk geographic area for GC development [129].
However, researchers concluded that in order to consider a direct role related to
these microbial composition variations in GC pathogenesis, further studies are necessary.
The colorectal cancer (CRC) represents the third most frequent cause of cancer-related
death in United States for both male and female, and its incidence has demonstrated
a growing trend in the last few years [130]. Natural history of CRC considers a sequence
of different anatomopathological entities that represent the stages through which
the transformation of normal mucosa cells in tumoral ones occurs. This process is
indissolubly related to genetic mutation acquisition, altered methylation of DNA,
modification of chromatin or altered expression of microRNAs [131]. Universally accepted
risk factors able to trigger this progression are age, type of diet (a diet rich in
red meat and low in fibers and fruits), obesity, tobacco, metabolic syndrome and intestinal
microbial composition modified by all the factors tested before [132]. A common point
that explains the mechanism that links these factors to CRC development is represented
by inflammation. In this regard, the role carried out by cyclo-oxygenase (COX)-2 and
inducible nitric oxide synthase (iNOS) that normally are not expressed in epithelial
cells and stromal cells is well known. COX-2 could have a role in determining the
progression of colon polyps in cancer by acting as procarcinogenic enzyme, through
the production of prostaglandin E2 (PGE2) that has proproliferative, proangiogenic,
and antiapoptotic properties [133]. Furthermore, COX-2 would be able to induce the
production of reactive aldehydes able to bind themselves and alter protein structure,
damage DNA or inhibit the reparation and promote the acquisition of essential genetic
mutations for disease progression [134]. In this intricate set of biological mechanisms,
in recent years, there has been room for research aimed at clarifying the role that
microbial intestinal flora could play in CRC despite the enormous difficulty in interpreting
most data from in vitro or animal models [135]. The presence of microbial biofilm
on the surface of colon adenomas has been recently found. It would be able to promote
the penetration of products of bacterial derivation into the superficial epithelium
through a decline of normal defenses of surface. The composition of microbial film
composition associated to colon adenomas consists of Bacteroidetes and Firmicutes
(Lachnospiraceae, Clostridium, Ruminococcus and Butyrivibrio) as well as Fusobacteria
and Gamma-proteobacteria. Moreover, it is clear that adjacent healthy mucosa is lacking
of typical biofilm found in correspondence of adenomas. According to the authors,
the correlation between biofilm and adenoma is to be researched in production by polyamines
bacteria that would be able to promote cellular growth and consequently CRC through
the induction of genotoxic damage [136].
Other microbial species (Fusobacteria) would be able to promote CRC through sulfide
production able to damage DNA. Fusobacterium nucleatum, for example, adheres, invades,
and induces oncogenic and inflammatory responses to stimulate growth of CRC cells
through its unique FadA adhesin. FadA binds to E-cadherin, activates β-catenin signaling,
and differentially regulates the inflammatory and oncogenic responses [137]. From
other studies, it was revealed that germ-free animals and animals treated with antibiotics
able to sterilize intestine had a reduced rate of tumoral development, even if these
results, in our opinion, complicate clinical evaluation given the impossibility to
apply experimental model used to humans [138], [139]. Moreover, this great approximation,
without an acute compositive evaluation of intestinal microbial species, on the role
of microbiota in CRC pathogenesis could lead to an incorrect information about this
topic.
Different microbial species carry out roles that are opposite in CRC. Just like Lactobacillus
and Bifidobacterium would have a role in prevention of cancer in animal models, a
manipulation of intestinal microbial composition carried out by nucleotide-binding
oligomerization domain-like receptor family members (e.g., NOD-2, NLRP, and IPAF)
would have a crucial role in bacterial eubiosis conservation [140], [141]. Loss of
either Nod2 or RIP2 resulted in a proinflammatory microenvironment that enhanced epithelial
dysplasia following chemically induced injury. The condition could be improved by
treatment with antibiotics or an anti–interleukin-6 receptor-neutralizing antibody.
Fecal microbiota transplantation from wild-type mouse to a knockout one for NOD-2
associates itself to a reduction of cancer risk, underlining a direct role linked
to intestinal microbial composition in genesis of this pathology [142]. IP degree
is another element to be taken into consideration in analysis of procancerogenous
stimuli supported by gut microbiota. Normally, PAMPs are able to overcome the filter
made up of surface mucus and cell tight junctions to reach submucosal level in contact
with innate and specific immunity cells. This type of interaction provides physiological
regulation of tolerance to microbial antigenes or their elimination through a process
of microbial scavenger that predicts the onset of inflammation. As long as this inflammatory
signal is activated in a “controlled” manner, it maintains bacterial eubiosis and
normal tissue renewal through the classical cell turnover. However, in condition of
IP increase as happens in pathological processes or particular diet habits, the dysregulation
of inflammation causes the activation of signal pathway that leads to cell proliferation,
as well as to be able to trigger DNA damage [143], [144].
In this regard, there are different studies in literature that demonstrate how an
IP increase is related to reduced elaboration of surface mucus and alteration of structural/reduced
expression of tight junctions is associated to a higher probability of developing
colitis, dysplasia and cancer [145], [146], [147]. The mechanisms that support cholic
cancerogenesis by gut microbiota are mainly related to induction of cell proliferation
as well as apoptosis inhibition. Enterococcus faecalis and Bacteroides fragilis would
be able, through COX-2 activation, to cause a proliferative effect related to superoxide
and activation of nuclear factor kappa B (NF-kB), respectively [148], [149] Some receptors
of innate immunity such as Toll-like receptors (TLRs) are able to recognize bacterial
antigenes and activate cell translation pathways that hesitate in triggering inflammation
and proliferative response. Normally, TLRs are insufficiently expressed in intestine,
and this expression, even in the liver, is inducible in relation to their activation
[150]. TLR-4 activation by lipopolysaccharides (LPS) induces proliferation through
COX-2 activation and epidermal growth factor receptor (EGFR) just like demonstrated
by a reduction of cell growth in TLR-4–deficient mice [151]. Moreover, TLR-4 activation
is able to induce angiogenesis, which represents a further crucial step for cell growth
and metastasis [152]. EGFR per se is able to promote cell proliferation through hydrogen
peroxide formation. Enterococcus faecalis is able to increase EGFR expression, favoring
this mechanism. Gefitinib use, EGFR inhibitor, is able to block Enterococcus faecalis–induced
cell proliferation [153].
On the other hand, enterotoxigenic Bacteroides fragilis, in a study in vitro, was
demonstrated to induce immediate apoptosis in HT-29 human colon cancer cells. However,
in a second observational experimental phase, the toxin-induced activation of p38-MAPK
and COX-2 was demonstrated to reduce apoptosis and promote cell proliferation [154].
Moreover, TNF-α activation, tumor necrosis factor receptor 2 (TNFR2) and NF-kB pathway
by Enterococcus faecalis are able to reduce apoptosis through an increase of netrin-1,
an important regulator of cell cycle [155]. Similar biological mechanisms able to
promote cancerogenesis were also taken into consideration in hepatocellular carcinoma
(HCC). It represents one of the most frequent neoplasias in the Western world since
about 78,000 new cases in United States by 2020 are estimated [156]. About 80% to
90% of HCC cases are consecutive to a chronic inflammatory damage related to liver
disease etiology (i.e., virus, alcohol, iron and copper accumulation), but it seems
to be connected to PAMPs arrival through portal blood. It is well described in literature
the role that gut microbiota plays in all phases of liver diseases. Indeed, it participates
in mantaining an inflammatory state that acts as driving force for progression to
fibrosis and cirrhosis [157]. The comprehension of connection between microbiota inflammation
and HCC is based on a few observations: a close vascular connection between intestine
and liver (gut-liver axis) exists. This axis receives nutrients not only by portal
blood but also by toxins released by gut microbiota in relation to IP levels. The
liver is rich in immunity cells (macrophages, lymphocytes, natural killer cells and
dendritic cells) that are able to respond to stimulus generated by PAMPs, with the
consequent induction of inflammation [158], [159]. The activation of inflammatory
signal focused on the activation of NF-kB–dependent signal pathway involves the production
of inflammatory cytokines such as TNF-α, IL-6, IL-1, ROS, etc., which, as we have
seen, are able to stimulate cell proliferation and inhibit apoptosis, although their
role is widely described in the promotion of the HCC rather than its induction [160],
[161], [162]. Hepatocarcinogenesis, as demonstrated by Dapito et al., would be related
to the activation by LPS of TLR-4 with the initiation of a chronic organ damage that
comes out in the promotion of proliferation at the expense of apoptosis by the production
of substances with mitogen activity like epiregulin [163]. The administration of antibiotics
in this regard would be able to turn off the proliferative signal mediated by the
activation of TLR-4, demonstrating an effective role of the microbiotic gut in the
progression of the HCC [164]. Inflammation and hepatocellular damage resulting thereof
are at the basis of the mechanisms for triggering a regenerative response. However,
this response would occur in nonphysiological conditions, supported also by the activation
and proliferation of stem cells (HSCs). They would contribute to the generation of
fibrosis and then liver cirrhosis. The microenvironment that is created leads to a
nonphysiological cell regeneration that is based on the deleterious damage-regeneration-repair
cycle. Such vicious cycle would be the basis of the loss of proliferative control
which may promote premalignant transformation and tumor growth [165]. In this regard,
HSCs would play an important role in controlling cell proliferation by acting as true
“mitotic controllers.” Such cells would be able to respond to stimuli by the presence
of surface receptors such as TLR-4, which are typically activated by ligands such
as LPS or desoxicolic acid [166]. The activation of these receptors involves the acquisition
of a senescence-associated secretory phenotype that favors the production of epiregulin
and therefore promotes cell proliferation and HCC progression. In a study by Yoshimoto
et al. correlation was made between the acquisition of senescence-associated secretory
phenotype of HSCs with the progression of HCC. Researchers have shown that mice with
high-fat diet, compared to those with conventional diet, were more likely to develop
HCC and had a greater density of senescent HSCs in peritumoral areas.
Enabling this functional profile to be acquired would be the inflammatory stimulus
dictated by the production of IL-1β, in turn produced for the signal triggered by
the high concentrations of desoxicolic acid and PAMPs in portal blood. In this sense,
HFD would be able to induce a change in the intestinal microbial composition, favoring
the survival of Firmicutes, especially Clostridium, which are capable of producing
high levels of desoxicolic acid from the metabolism of colic acid. Such link ring
explains the increased activation of NF-kB in mice fed with HFD and provides a basis
for understanding the role of HSC in hepatocyte cancer [167].
Finally, pancreatic cancer is one of the most aggressive neoplasms, with a mortality
rate that can reach over 90% of new cases within the first year of diagnosis [168].
For this reason, the understanding of the pathophysiologic mechanisms responsible
for its onset and/or its progression is the basis, together with an appropriate renewal
of therapies, to reduce the social impact of this disease. Again, chronic inflammation
is the basis of the pathophysiological process that supports the formation of pancreatic
cancer. Although the pancreas, unlike the liver, does not exhibit its intrinisc microbiome,
according to a recent scientific theory, it could be reached by PAMPs both in the
circulatory stream and through the biliary/pancreatic tract (transductal transmission).
The arrival of such antigens leads to stimulation of innate immunity receptors, including
TLR-4, resulting in proinflammatory cytokine production that would act along with
other risk factors such as obesity and smoking for the onset of the disease [169].
The combination of the classic risk factors for pancreatic cancer and the penetration
of PAMPs into the portal circle is evident in alcohol abuse. Alcohol is, in fact,
a risk factor for pancreatic disease, from pancreatitis to cancer, but is also one
of the best known causes of increased bowel permeability, leading to circulatory delivery
of bacterial-derived products [104].
Such interesting and promising scientific discoveries are paving the way for a better
understanding of pathogenetic mechanisms that support gastrointestinal cancer, enabling
the scientific community to emerge on a new scenario that is promising to be exploited
as a new therapeutic weapon against such diseases. However, nowadays, the results
obtained from in vitro and experimental animals are not applicable to biology of human
systems and therefore require further confirmations and in-depth studies. The key
point for application to human therapy may be from the possibility in the near future
of selective or superselective manipulation of intestinal microbial species, enabling
a specific “microbial personalization” for the specific patient.
Conclusion
For several years, scientific evidence has shown the link between inflammation and
cancer. In this context, it is clear that an inflammatory microenvironment is an essential
assumption for the development of most tumors. Each cell and the proportion of specific
inflammatory cell types in tumoral microenvironment could have a polarized role in
tumor progression or tumor suppression according to the production of autocrine, paracrine
and endocrine substances.
Moreover, inflammation is able to determine the production of angiogenetic factors
and to promote the survival of tumor cells through a direct route, that is, the increase
in oxygen supply and nutrients to tumor tissue, and an indirect route, which increases
the recruitment of proinflammatory cells and releases cytokines that promote growth,
invasion of tumor metastasis.
Several chronic inflammatory diseases are also able to determine an increase of cancer
risk as happens in patients affected by IBD or COPD. In these chronic inflammations,
in addition to the cytokine proinflammatory effects, several molecular alterations
are also involved in the inflammation-induced carcinogenesis, such as inactivation
of tumor-suppressor genes, oncogene mutations, loss of heterozygosity, and chromosomal
and microsatellite instability.
A common mediator of carcinogenesis in inflamed tissues is the imbalance of oxidative
stress induced by inflammation in a normal tissue and sustained by microenviromental
inflammation in a context of malignant tumor.
Another factor involved in the generation of chronic inflammation that sustains cancer
is represented by endotoxemia. The communication between “gut microbiota system” and
liver, brain, adipose tissue and other organs of human body is regulated by intestinal
permeability (IP). Its increase, in relation to a dysbiotic gut, can be considered
a key point for the comprehension of some pathogenetic mechanisms of systemic and
liver disorders as well as cancer. Indeed, there is a relationship between IP and
plasma levels of proinflammatory cytokines, nitric oxide, and nitrite levels in patients
with various types and degrees of chronic liver diseases.
However, despite the growing interest regarding this field, nowadays, a standardized
protocol to study the relationship between gut microbiota and cancer especially in
the interpretation of study results does not exist. The strongest scientific evidence
of this relationship exists in relation to tumors of gastrointestinal tract. For this
reason, we focused our attention on the pathogenetic mechanisms sustained by gut-derived
inflammation in the development of gastrointestinal cancer.
Bearing in mind these mechanisms that link inflammation, oxidative stress and IP to
cancer, it could be useful to plan the prophylaxis and the therapy of cancer in the
next future.