Due to the overwhelming burden of colorectal cancer (CRC), great effort has been placed on identifying genetic mutations that contribute to disease development and progression. One of the most studied polymorphisms that could potentially increase susceptibility to CRC involves the nucleotide-binding and oligomerization-domain containing 2 (NOD2) gene. There is growing evidence that the biological activity of NOD2 is far greater than previously thought and a link with intestinal microbiota and mucosal immunity is increasingly sought after. In fact, microbial composition may be an important contributor not only to inflammatory bowel diseases (IBD) but also to CRC. Recent studies have showed that deficient NOD2 function confers a communicable risk of colitis and CRC. Despite the evidence from experimental models, population-based studies that tried to link certain NOD2 polymorphisms and an increase in CRC risk have been described as conflicting. Significant geographic discrepancies in the frequency of such polymorphisms and different interpretations of the results may have limited the conclusions of those studies. Since being first associated to IBD and CRC, our understanding of the role of this gene has come a long way, and it is tempting to postulate that it may contribute to identify individuals with susceptible genetic background that may benefit from early CRC screening programs or in predicting response to current therapeutic tools. The aim of this review is to clarify the status quo of NOD2 mutations as genetic risk factors to chronic inflammation and ultimately to CRC. The use of NOD2 as a predictor of certain phenotypic characteristics of the disease will be analyzed as well.

Despite several advances in the diagnosis and treatment of colorectal cancer (CRC), it continues to be one of the most significant causes of morbidity and cancer-related deaths[1]. Apart from familial syndromes that account for 5%-10% of CRC cases[2], this disease is considered to have a multifactorial etiology and therefore predicting individual risk has been problematic, as there are many genetic polymorphisms with probably modest individual effect[3]. Due to its proven role as a genetic predisposing factor for chronic inflammation, most notably in Crohn’s disease (CD)[4], nucleotide-binding and oligomerization-domain containing 2 (NOD2) mutations have been suggested to have a similar role in CRC. This assumption derives from the fact that several gastrointestinal cancers are strongly linked to chronic inflammatory conditions. The risk for malignancy may even increase according to the degree of underlying inflammation, as is the case for long-standing ulcerative colitis[5] and Helicobacter pylori (H. pylori)-induced chronic gastritis. One plausible hypothesis is that pro-inflammatory stimulus may lead to continuous cell proliferation, angiogenesis and eventually DNA damage[6]. If the association between chronic inflammation and cancer is based on solid experimental and epidemiological data, what remains to be fully understood is how do NOD2 mutations lead to chronic inflammation?

The NOD2 protein plays an important role in innate immunity by recognizing bacterial lipopolysaccharides and activating the nuclear factor-kappaB. The mutant alleles for NOD2 gene are thought to cause loss of function - deficient recognition, impaired clearance and proliferation of bacterial pathogens that lead to increased pro-inflammatory cytokines and subsequently to chronic inflammation[7]. Recently, the role of NOD2 has been analyzed under a different light, as it seems to take a central place in the intricate balance between protection of the intestinal mucosa under physiological settings and the production of pro-inflammatory cytokines in chronic inflammatory conditions. Any shift, either gain or loss of function for NOD2, elicits a disturbance in the immune system that may lead to inflammation[8].

Another promising approach to understanding the pathogenesis of these diseases is the modulation of intestinal microbiota. Formerly considered a passive element in the homeostasis of the intestinal mucosa, the microbiota is nowadays considered as essential for epithelial differentiation and in maintaining a protective environment[9]. To that end, commensal bacteria and NOD2 interact in a feedback-like mechanism - NOD2 keeps bacterial proliferation in a steady, controlled state and the microbiota intervene in controlling NOD2 expression[10]. Animal models with high risk genotype for colitis and CRC show less severe or even absent inflammation and fewer cases of adenomas if raised in selected bacterial or germ-free conditions[11], suggesting that changes in microbial species affect colitis and CRC development.

From 2004 to 2010, several population-based studies tried to find an increased risk for CRC in NOD2 polymorphism carriers. The most studied mutations have been two missense mutations - R702W (rs2066844 C/T) and G908R (rs2066845 C/G), and a frameshift mutation - 3020insC (rs2066847 insC). The frequency of these polymorphisms differs greatly between populations, being much less common in Asian cohorts[12]. Even among Caucasian populations, there is significant genetic heterogeneity that may have limited the findings of such studies. Furthermore, the source of controls and the type of methodology used are also potential bias. Another relevant finding is that individual polymorphisms may increase the risk of CRC only in selected groups (e.g., German individuals under 45-year-old carrying R702W polymorphism[13]). These promising studies may ultimately lead to identifying individuals with susceptible genetic background, other than those with well-known familial syndromes, therefore benefitting from early diagnostic screening.

In order to find recent publications (2004 to present) on NOD2 mutations and CRC susceptibility, an extensive literature search was performed using PubMed and MEDLINE. The key search terms used were NOD2/caspase recruitment domain 15 (CARD15) mutations, CRC genetics, microbiota, mucosal immunity and chronic inflammation, either alone or in combination. All articles identified were English-language, full-text papers. We also searched the reference lists of identified articles for further relevant papers.

In order to protect the epithelium of the gastrointestinal tract, the largest surface in our body exposed to external environment, a number of obstacles within the mucosa are disposed to prevent the spread of pathogenic organisms. Besides mechanical barriers such as the mucus layers, the innate immune system is probably the first mechanism to act against deleterious microorganisms, and once triggered it becomes activated within minutes[14]. This almost immediate response is due to pathogen recognition receptors (PRRs). One of the most prominent families of PRR includes Nod-like receptors (NLRs) that recognize bacterial wall component peptidoglycan. Certainly the most researched member of this family is NOD2, expressed in dendritic cells, leukocytes and epithelial cells of the gastrointestinal tract, especially after inflammatory stimuli[15]. Paneth cells are secretory epithelial cells found in small bowel crypts that express significant levels of NOD2. The structural features of NLRs include a central nucleotide-binding oligomerization domain, a variable N-terminal protein-protein interaction domain, defined by the CARD, and a C-terminal leucine-rich repeat that senses pathogen-associated molecular patterns[8]. After activation, recruitment of a serine threonine kinase called RIP2 occurs, which leads to the enrollment of the NF-κB signaling pathway, leading to the transcription of immune response genes[16].

Along with this traditional role, NOD2 plays a part in the induction of autophagy as well. This process leads to the destruction of damaged proteins and organelles which has paramount importance not only in recycling biomolecules but also in eliciting anti-microbial properties[17]. Growing evidence has shown that NOD2 may be also a relevant player in CD4⁺ T cell function and in generating a Th1 response. This leads to the production of IFN-γ by T cells. In addition to detecting bacterial pathogens, NOD2 has shown a different role in host defense. According to in vitro studies, NOD2-deficient animals showed an increased susceptibility to several viral infections[18]. The response to certain parasite infections may be compromised as well, due to a reduced production of IFN-γ[16,19]. In NOD2^-/- animals, through stimulation of Th1-associated cytokines, an increase in mucosal permeability and low-grade chronic inflammation occurs[20].

The aforementioned evidence places NOD2 in the center of the immune system, quite distant from its first described role as a “simple” pathogen sensor. But the essential question remains: How do NOD2 mutations predispose to CD and do they have a role in cancer development?

Although our understanding of these complex interactions has improved in recent years, we still have no solid evidence to prove that polymorphisms in NOD2 actually lead to the production of a stable protein. Currently, there are at least two possible ways in which NOD2 mutations can lead to chronic inflammation and CD. The first is related with the basic function of NOD2 as a positive regulator for innate response. We can assume that if there is no efficient NOD2 activation during early phases of pathogen exposure, bacteria will proliferate and ultimately lead to chronic inflammation. An alternative explanation involves the deficient activation of NOD2 as well, but in a later stage, during an ongoing inflammatory process. A continuous stimulus of pro-inflammatory pathways will take place, triggering toll-like receptors (TLR) (NOD2 is a negative regulator of TLR signaling) and favoring Th1 response and the release of cytokines. Animal models have shown that NOD2 has a paramount importance in the bactericidal activity of ileal crypts and the regulation of ileal microbiota. This allows us to hypothesize that ileal CD caused by NOD2 mutations is due to the dysfunction of Paneth cells[8,10,21]. This diverse function of NOD2 may explain the heterogeneity of CD[16].

In what concerns CRC, there are several factors that could explain its link to NOD2 mutations. It is a well-known fact that the risk for CRC increases with duration and severity of the inflammatory process, while it decreases when anti-inflammatory drugs such as mesalazine and immunomodulators such as azathioprine are used in ulcerative colitis, consistent with a causative role for inflammation in colon carcinogenesis. The chemopreventive activity of aspirin and other nonsteroidal anti-inflammatory drugs in CRC supports this concept as well.

If these mutations play a consistent part in the pathogenesis of inflammatory bowel diseases (IBD), most notably in CD, then the hypothesis that such mutations are potential risk factors for cancer may have solid grounds. The study by Couturier-Maillard[9] shed some light in the role of NOD2 in colitis-associated cancer: NOD2^-/- mice showed increased tumor load in the distal colon than wild-type animals. Furthermore, this risk was shown to be transmittable if both animals were cohoused. An acceptable rationale to explain these findings may involve an unbalance between pro- and anti-inflammatory cytokines and lead to the loss of autophagy and apoptosis stimuli. This could eventually lead to increased risk of infection, chronic inflammation and cancer[22].

The most promising body of evidence that supports the role of NOD2 in colorectal carcinogenesis involves its capacity to shape a protective assembly of gut bacterial communities. The deregulation of intestinal microbiota seems to be the essential element in the complex interaction between NOD2 mutations and CRC.

Recently, a significant effort has been made to identify the components and the role of intestinal microbiota in colonic health and homeostasis. The microbiota has been increasingly recognized as a major player in normal metabolic and physiologic processes. This community of 10¹³-10¹⁴ microorganisms represents a perfect example of symbiotic relationship. It exerts several essential functions like the synthesis of essential compounds for the normal growth of colonic mucosa, regulation of its lymphoid tissue and synthesis of amino acids that inhibit the growth of pathogenic microorganisms. The latter is of paramount importance, as a delicate equilibrium has to be achieved between commensal bacterial load and the innate immune system. The commensal bacteria must be contained in their capacity to proliferate and should be maintained in adequate amounts. Conversely, any change in the composition of luminal bacterial flora may favor the production of toxins and metabolites associated with carcinogenesis and induce dysregulation of the immune response, allowing pathogenic agents to replicate, therefore promoting and sustaining inflammation and carcinogenesis[23]. A well balanced gut microbiota leads to healthy colocytes through the production of important compounds and the correct modulation of the immune system. It is now believed that NOD2 mutations may result in altered host-microbial interactions in the intestinal mucosa.

It is known that the human intestine is sterile in utero, but progressively over 36000 bacterial species colonize the gastrointestinal tract and develop a symbiotic relation with their host. The human distal intestinal microbiota includes two predominant phyla, the Firmicutes and Bacteroidetes, with less significant contributions from Proteobacteria and Actinobacteria, and minor contributions from Fusobacteria, Verrucomicrobia, and Cyanobacteria[24]. The production of propionate and butyrate from the degradation of indigestible polysaccharides is one of its main roles. Despite being outnumbered by other species, Actinobacteria, Proteobacteria (including Escherichia coli) and Verrucomicrobia, showed potential to influence health outcomes[25]. A significant effort is being made to characterize the diverse genetic material of these numerous microorganisms (also known as microbiome). Ultimately, the goal is to understand the link between variations in the composition of these communities and common diseases, such as IBD and CRC. The hypothesis formulated by several investigators lies on the ability of these variations of the microbiota to cause a breakdown of the balance between bacterial communities and the epithelial barrier may lead to chronic inflammation. It is thought that some species may not be able to maintain a quiet state of protective immunity in dysbiotic conditions. For example, Bacteroides thetaiotaomicron was found to be not only a commensal but also an opportunistic microorganism in predisposed individuals[26].

A special attention has been devoted to factors that may influence the composition of the microbiota. Aging, place of birth and mode of delivery are quite relevant determinants. Antibiotics are also important and modifiable factors, especially with larger antimicrobial spectrums, as their effect on the microbiota varies from drastic to only temporary. After the end of the antibiotic treatment, certain species may recover in about a month while others may not recover at all, most often in children[27]. Besides antibiotics, different dietary habits may modify significantly the microbiota as well. Quite interestingly, even a short-term increase in fat and carbohydrate consumption may influence not only the relative abundance of each bacterial species, but also the functionality of the microbiota[28]. Obesity has also been proposed as a major factor in this equation, as it may alter the composition of the microbiota and increase its metabolic potential to harvest energy from the host diet. The capacity of the microbiota to confer host traits was revealed by studies where fecal content was transplanted from obese mice into lean germ-free recipients. The recipients showed significant weight gain and increased adiposity[29]. This surprising discovery opened the door for further investigation on genetic and microbiota manipulation and its ability to cause disease in animal models. Another interesting concept was revealed by Couturier-Maillard et al[9]. The absence of NOD2 confers a transmissible risk for colitis and CRC, even to immunocompetent hosts. In other words, after sharing the same environment with NOD2^-/- mice, wild-type animals treated with dextran sodium sulfate revealed an increased risk for colitis, probably due to an altered commensal flora acquired from knockout mice. This dysbiotic microbiota is then passed to the next generation. Growing evidence now shows us that the risk for colitis and CRC is influenced by specific members of the commensal microbiota. In fact, in IL10-deficient mice treated with colon-specific carcinogen azoxymethane (AOM) showed high levels of mucosal inflammation and adenoma development when raised in conventional conditions, but when Bacteroides vulgatus is the only commensal, the carcinogenicity of AOM is somewhat attenuated. Even more surprising is the fact that these mice show almost no inflammation or adenomas if created in a germ-free environment[11]. This is consistent with the notion that treating NOD2-deficient mice with broad spectrum antibiotics may mitigate its disease risk[9]. In the opposite direction, a recent study suggests that antibiotics promote inflammation through translocation of commensal colonic bacteria and it is suggested that this may explain the association between increasing antibiotic use and the growing incidence of inflammatory disorders[30].

The importance of NOD2 as a regulator of microbiota and consequently as a risk factor for injury of the colonic mucosa was reinforced by studies using reciprocal fecal microbiota transplantation. Such interventions led to profound changes in the microbiota. The end result was that NOD2-deficient mice that received fecal transplantation from wild-type animals showed decreased mucosal injury and inflammation. On the contrary, there was an increased risk of colonic disease in wild-type hosts that received dysbiotic fecal microbiota from NOD2-deficient mice[9]. A crucial role in this equilibrium is played by Paneth cells, specialized secretory epithelial cells of the small intestinal crypts that express NOD2 at high levels. Recent studies demonstrated that NOD2-deficient mice had a significant rise in the amount of commensal bacteria in the terminal ileum, probably due to impaired cryptal activity[31]. This is a potential mechanism by which NOD2 mutations may disturb intestinal homeostasis and lead to CD and colitis-associated CRC. But the intestinal microbiota role in this equilibrium is not passive at all. On the contrary, it is now known that it plays a part in controlling the expression of NOD2 as well.

The influence of NOD2 in microbial communities and its consequences on disease risk have solid basis. But how exactly does an altered microbiota lead to colonic inflammation? One possible explanation is the production of bioproducts with anticancer properties by the metabolic machinery of the microbiota. Butyrate and other small-chain fatty acids are nutrients formed by the fermentation of indigestible carbohydrate and are known to have an interesting paradoxal activity. In colon carcinoma cells, it leads to apoptosis, inhibits cell proliferation and angiogenesis, therefore showing a protective effect. On the other hand, in normal colonic cells it shows opposite effects as it prevents apoptosis[32]. Another potential role for the microbiota and especially for butyrate is maintaining inhibition of the histone deacetylase, therefore maintaining histones in an acetylated state, thus facilitating the transcription of anti-oncogenes[33]. This is an objective of anticancer drugs such as Vorinostat, an approved agent for the treatment of cutaneous T cell lymphoma. Detrimental influences are provided by the accumulation of toxic compounds in the gut that can exert a mutagenic action. Poli-heterocyclic amines, deoxycholic acid and calibactin are examples of compounds directly or indirectly produced by commensal bacteria that harbor potential to damage colonocytes’ DNA[34]. This conflicting evidence supports the idea that the role of the microbiota shouldn’t be considered univocal, but rather be regarded as a complex set of influences that may have a protective or deleterious effect on mucosal immunity, depending on its specific components. It is reasonable to postulate that the same bacterial agents will elicit different effects according to each individual genetic background and environmental exposure.

Since the publication in 2001 by Ogura et al[4] of the first study about NOD2 mutations and increased susceptibility to CD, several investigators tried to understand the intricate mechanism behind this association and to find a link between these polymorphisms and other inflammatory and malignant conditions.

In fact, a role for NOD2 mutations has been postulated for several malignant diseases other than CRC. In common digestive tract tumors, a significant number of published studies have addressed this putative relation. The 3020insC missense mutation was shown to be a risk factor for intestinal type gastric cancer in a Portuguese population[46]. The same was demonstrated by an Italian study for the R702W and 1007fs polymorphisms[47]. According to the meta-analysis by Liu et al[37], there was an increased risk for gastric cancer for carriers of the G908R and 3020insC mutations, but the same was not observed for the R702W polymorphism. In a recent population-based Chinese study, an increased risk for gastric cancer was found for individuals carrying the rs718226 AG or GG genotype. Interestingly, this single-nucleotide polymorphism revealed significant joint effects with H. pylori in dysplasia and gastric cancer risk. On the contrary, both the rs2111235 C allele and the rs7205423 G allele showed a protective effect, as they were associated with a decreased risk of progression to dysplasia and gastric cancer in H. pylori-infected subjects[48].

In what concerns pancreatic cancer, there was no evidence of increased risk neither in the familial nor the sporadic form of the disease[37,49]. The same meta-analysis revealed an increased risk for MALT lymphoma, breast, lung and laryngeal cancer for the carriers of the 3020insC mutation. On the other hand, none of the NOD2 mutations were found to be risk factors for melanoma or non-Hodgkin lymphoma[37].

These discrepancies found between different studies may be attributed to a variety of factors, especially those that influence the expression of these polymorphisms, as well as differences in sample size, geographic variation or genotyping methods[22].

The effect of NOD2 as a risk factor for disease is best established in CD. After an etiologic role was consolidated for these mutations in CD, further investigation was undertaken to find out if these mutations influenced the behavior, prognosis and response to treatment as well. The presence of one mutation increased the risk for structuring or penetrating disease by 8% and this effect was largely increased if two NOD2 mutations were present (41% risk increase)[38]. An Australian study revealed as well that carriers have a more aggressive disease, needing more frequent and more precocious surgery[50]. A recent European multicenter cohort study recently revealed that NOD2 mutations and early use of immunomodulatory drugs are the most relevant predictors of the course of disease[51].

It was speculated as well that certain disease phenotypes and their response to treatment could be influenced by NOD2 mutations. The development of perianal fistulas is thought to depend on the proliferation of luminal bacteria. As such a possible connection between NOD2, a regulator of host response to microbial agents, and perianal fistulas was evaluated in recent literature. These fistulas showed significantly worse response to antibiotics in NOD2 mutation carriers[52], probably due to impaired recognition of intestinal bacteria and a decreased ability to mount an effective innate immune response. This kind of studies emphasizes the importance of gene mapping and corresponding phenotypic correlations in order to predict disease severity and optimize treatment strategies.

In the last fifteen years, the proposed role of NOD2 and its mutations in disease has grown significantly. From only a susceptibility gene to an important predictor of prognosis and response to treatment in CD, these mutations have been postulated as a risk factor in several conditions such as mycobacterial infections, common gastroenterological disorders and malignant diseases like gastric and colorectal cancer. The expanding role of this receptor as a major coordinator of several inflammatory pathways and a modulator of microbiota is increasingly accepted, mainly due to evidence arising from NOD2-deficient animal models. In fact, it was shown that losing NOD2 activity leads to more severe colitis and higher propensity to adenomas and CRC. It seems likely that NOD2 may be the key element of the intricate puzzle that links the disturbance of mucosal immune defense, dysbiotic bacterial communities and conditions such as CD and colitis-associated CRC.

Furthermore, NOD2 polymorphisms such as 3020insC and R702W seem to increase susceptibility to CRC. The search of these mutations is still not offered routinely in clinical practice. However, the identification of its carriers would allow such patients to be included in a more intense CRC surveillance program, contributing to early diagnosis of a disease that carries such a heavy burden. Predicting response to different chemotherapy regimens according to the presence of NOD2 polymorphisms could become a useful tool for clinicians. More large-scale studies should be conducted to confirm this association. The development of new therapeutic targets based on research about NOD2 protein function and interactions could ultimately lead to a tailored approach to the treatment of CRC.