Microbes, Microbiota and Colon Cancer

Introduction

The human large bowel is a common site for adenocarcinomas and also one of the most densely populated microbial ecosystems on our planet. Colorectal cancers (CRC) affect over a quarter of a million people each year. In industrialized nations, the lifetime risk of developing CRC is approximately 5% and the lifetime risk of developing an adenoma, a non-cancerous colon tumor that can develop into CRC, is 20%. When the disease is local or confined cure rates range from 70–90%, however, advanced colorectal cancer has a high mortality rate, consistently ranking in the top 3 causes of cancer-related death around the globe. There has been long standing curiosity about the role of bacteria in colorectal carcinogenesis, because of the large disease burden of CRC and the microbial load of the colon; and recent heightened interest in the gut microbiome in CRC, because of the increasing accessibility of microbial meta’omics.

Sequencing technologies have vastly expanded our understanding of the human genetic landscape of colorectal cancer. Similarly, efforts at sequencing CRC microbiomes are providing leads into how a microbe’s interactions with an individual’s entire colonic microbial community, clades within that community, or the human holobiont (Gordon et al 2013), the entirety of the assemblage of both human and microbe, may be associated with colorectal carcinogenesis. Studies of candidate species in model systems have been useful in evaluating cancer causality and are in keeping with reductionist scientific experimental paradigms. However, an equally plausible concept is that consortia of microbes contribute to colorectal cancer risk over time, which can be a far more challenging concept to observationally or experimentally interrogate. This concept is well-aligned with human genetic-based models of colorectal carcinogenesis, namely that, molecular alterations in multiple genes underlie the development of a hyperplastic epithelium and propel progression onto adenoma and then towards adenocarcinoma. Mutations in human genes that influence adenoma and adenocarcinoma development may shape the growth rate of colonic epithelial cells, reduce their susceptibility to cell death, endow them with metabolic specializations, and confer on them abilities to commandeer immune cells to further promote growth and spread. Similarly, microbes can be viewed as collections of gene networks that affect cancer genomic stability, metabolism, and immune responsiveness. In turn, it is possible that the characteristics of transformed colonic epithelial cells render them more sensitive to microbially-influenced carcinogenesis.

Herein, we will discuss recent insights into contributions of the microbiota to CRC. We explore conceptual frameworks for evaluating the role of microbes in cancer causation as we highlight new findings on candidate CRC-potentiating species and knowledge gaps. We will not summarize how microbially-elicited inflammation or host microbial-sensing pathways affect carcinogenesis as these topics have been the subject of several recent reviews (Dejea et al., 2013; Kostic et al., 2013; Jobin, 2012; Schwabe and Jobin, 2013 and see review by Trinchieri and Goldzmid in this issue). Instead, we concentrate on microbial metabolism in colorectal carcinogenesis with a focus on bile acids and also touch upon xenobiotics and food, all of which are areas where the microbiota has the potential to explicate observations about host gene-environmental interactions in carcinogenesis.

Causality Theory

CRC is essentially a genetic disease (Figure 1). Gradual accumulation of oncogenic gene mutations leads to autonomous colonic epithelial cell (CEC) proliferation that slowly progresses typically over 10 to 40 years, resulting in first colon adenomas then, in a minority of individuals, cancers. Initiation of colon tumors (adenomas, adenocarcinomas) refers to events yielding biologic changes fostering CEC proliferation; progression refers to the subsequent events that liberate growth of the incipient colon tumor and, ultimately, transformation to cancer. Yet, it remains unknown with any precision what events precipitate either the initial, disease-initiating mutation(s) or foster the subsequent disease progression. The microbiome, however, is a prime suspect for triggering the initiation and/or progression of colonic carcinogenesis. Certainly, in murine disease models, colon mucosal inflammation, often induced by mucosal irritants (dextran sulfate sodium, 2,4,6-trinitrobenzenesulfonic acid) combined with administration of a carcinogen (often azoxymethane, a compound in engine fuel), yields colon tumorigenesis, substantiating the intersection of inflammation and exposure to carcinogens in colon tumorigenesis. Certain engineered murine gene knockouts, with potential mucosal inflammation sequelae, also yield colon carcinogenesis that is ameliorated in germ-free animals or sometimes merely by a vivarium change, often considered a proxy for acquisition of a new microbiota. While these models support the hypothesis that the microbiota contributes to colon carcinogenesis, they mimic human disease development poorly. Until recently, the contribution of the human colon environment, home to the largest and most complex microbial mass of human ecology, was not integral to the analytical framework of translational CRC research.

What can guide us as we seek to determine if and how the colon microbiome contributes to the pathogenesis of sporadic human CRC? One clear limitation in seeking a microbe as the cause of a chronic disease is the possibility that the inciting microbe is no longer present at the time the disease is identified, perhaps gradually eliminated by changes in tumor microenvironment no longer hospitable to the microbe or, alternatively, because the microbe acts by a ‘hit and run’ mechanism whereby limited microbial exposure is sufficient to incite disease. For example, in the case of H. pylori and gastric cancer, isolation of H. pylori declines with advancing gastric cancer although detection of H. pylori exposure is usually still possible by serology (Ota H et al., 1998). This reinforces the importance of utilizing multiple approaches in seeking to link a microbe to disease ‘causation’.

Figure 2 provides a framework for considering the microbiota and specific members of the microbiota as either primary (initiators) or secondary (fostering progression) contributors to human CRC pathogenesis (Sears and Pardoll, 2011). We consider 3 models by which specific microbes (Model 1), a microbial community (Model 2) or the two acting sequentially and/or in synergy (Model 3) influence colon carcinogenesis. With respect to the first model, we well understand that individual microbes such as the pneumococcus, the meningococcus, Helicobacter pylori or hepatitis viruses are established etiologies of host pathology. We consider that these microbial pathogens possess sufficient virulence mechanisms enabling them to act alone in disease causation. In contrast, as a second model, inflammatory bowel disease stands as the best prototype for microbial community disease causation (Sears and Pardoll, 2011). In this disease, host genetics is the presumed initiator permissive to development of a dysbiotic (implying dysfunctional, disease-initiating or -amplifying) microbiome with an ensuing cycle of host gene-microbiota interactions causing intestinal and possibly extraintestinal disease. Experimental work definitively supports this pathogenetic sequence in that dysbiotic colonic microbiota develop in, at least select, mice with gene knockouts (e.g., Tlr5, Il10, T-bet and Rag2) and that this emergent dysbiotic microbiome alone possesses the capacity to transmit to a healthy mouse (without any gene mutations) the disease of interest (Garrett et al., 2007, 2010; Vijay-Kumar et al., 2010). Importantly, these murine experiments strongly support the idea that the dysbiotic microbiota can, as a community, encode tissue-specific (e.g., colitis) as well as systemic disease (e.g., metabolic syndrome, obesity) (Ridaura et al., 2013; Vijay-Kumar et al., 2010).

Human CRC provides an opportunity to consider individually the above disease causation scenarios as well as a novel hypothesis in which these theories blend with individual or limited bacterial species acting in concert with a locally modified microbiome to cause CRC (Figure 2, Model 3). Nuances of a limited microbial consortium inducing colon tumorigenesis include the possibilities of sequential microbial exposure or polymicrobial disease causation. For this third disease model, we lack clear, clinically relevant historical examples to guide us except for the requirement of the hepatitis B virus for replication and disease induction by the hepatitis D virus. For example, while we understand that the pneumococcus or the meningococcus invade the host from among a complex microbiota in the respiratory tree or nasopharynx, we have no clear data suggesting that the composition of these microbial communities are required for or contribute to the disease causation potential of these bacteria. Similarly, the intestinal microbiota has been shown to hinder or exacerbate viral infections in murine models but the responsible bacteria are not known (Wilks et al., 2013).

It will not be easy to discern among these potential disease models that provide a framework for defining the microbe contributions to human CRC pathogenesis. Carefully designed studies that consider the Bradford-Hill criteria (1965) are needed to link the microbiota and/or select microbes with CRC initiation and progression. A view from the colon lumen may be insufficient. Rather, microbial data must be considered in the context of key host parameters such as the host immunologic response (including the tumor microenvironment) and host gene polymorphisms that influence the host immune response as well as host susceptibility to CEC gene mutations.

The Microbiome Community as Protagonist

The seminal work of Eckburg et al. clarified the complexity of the fecal and mucosal colon microbiota, importantly illustrating two key points relevant to the colon microbiome as causal in colonic carcinogenesis (Eckburg et al. 2005). First, the majority of microbes, predominantly bacteria, within the colon microbial community are ‘non-cultivatible’. While this concept has been challenged by subsequent work, it remains clear that a complete cultured or genome sequence catalog of the colon microbiome with strain-level resolution is still far from our reach (Lagier et al., 2012). Thus, at this time, associations between the microbiota and CRC rely on approaches to broadly define the composition or function of the colon microbiome using various ‘omic’ approaches (16S rRNA gene sequencing, metagenomics, transcriptomics, proteomics, metabolomics). Second, while the colon mucosal community may not vary substantially along the axis of the colon, the mucosa-associated community differs from the intraluminal microbiome. These data raise the important and yet unanswered question of whether the fecal microbiome alone will sufficiently mirror mucosal events to allow ‘causation’ to be established.

The dataset to address microbial community associations in human CRC is limited and has focused on defining bacterial communities associated with colon tumorigenesis. The available studies yield several worthy, albeit preliminary, observations (Dejea et al., 2013). First, the bacterial community composition in colon adenoma or CRC patients, both in mucosal samples and feces, differs from the examined control samples although consistent associations of bacterial groups with tumor samples or tumor hosts is not yet discernible. Second, ‘on tumor’ and ‘off tumor’ mucosa bacterial populations differ within the tumor host. Third, the microbiota of CRC tissue samples and luminal samples differ consistent with prior studies in healthy individuals. Lastly, while considerable study-to-study differences in detected bacterial groups by sample are reported (including nearly polar opposite findings), studies have identified enrichment of Fusobacterium spp. (most often identified as F. nucleatum) associated with CRC mucosa compared to non-tumor, histologically normal colon tissues from the same cancer-bearing host (Castellarin et al., 2012; Kostic et al., 2012). Additional work has further identified enrichment of Fusobacterium spp. in fecal samples from CRC hosts compared to healthy individuals (Ahn et al., 2013; Kostic et al., 2013). These observations are discussed in more detail below. Several of the available colon tumor microbiome studies have limitations including small sample sizes, undefined tissue sampling sites, limited or absence of control samples (including healthy controls and/or control tissues within the tumor host), limited or no metadata (subject clinical data, e.g. patient age, family history, past medical history, current and past medications, dietary patterns, cancer stage, or tumor KRAS, PIK3CA, BRAF, NRAS mutational status) and/or poorly described analyses. Differing study designs including types of samples analyzed and populations included combined with, for example, unknown dietary habits or other risk factors for CRC that are associated in other datasets with modified colon microbiomes, make it difficult to glean themes across these studies.

When considered within the Bradford-Hill framework for establishing causality, our knowledge of the community microbial associations in CRC remains quite limited. For example, as yet, no study has examined in parallel fecal and mucosal samples from non-tumor hosts compared to the fecal and mucosal communities present in colon tumor hosts (‘on-tumor’ and ‘off-tumor’). This approach might help us discern specific microbial associations and could provide the basis for prospective testing for disease identification. Prospective, longitudinal study of individuals at high risk for development of CRC will need to be done to discern if shifts of the colon microbiome occur coincident with disease development consistent with the epidemiologic principle that exposure must antecede disease expression. Serologic, proteomic or metabolomic studies of human CRC and controls to identify potential fecal, mucosal or serum colon tumor specific molecular markers for disease prediction that might simplify screening for this highly preventable disease are also outstanding. Further, although the right and left colonic mucosal communities appear similar in the healthy host, the molecular features of right and left CRC can differ (Yamauchi et al., 2012), raising the question of whether regional microbial associations may distinctly affect the genesis of colon tumors, a point not yet addressed by available data. Lastly, there is uncertainty about the population sizes required to identify the ‘correct’ causal associations, if these exist. For example, the population size to detect a single microbe as causal will likely be much smaller than if a particular community exposure or polymicrobial, even sequential, exposure is critical. Certainly, large populations were necessary to identify the effect of KRAS mutations, aspirin or diet on colorectal carcinogenesis.

Individual Microbes as Protagonists

There is a long history of attempting to associate individual microbes, mostly bacteria, with human CRC (Aries et al., 1969; Hill et al., 1971). As examples to guide our thinking, herein we discuss data on select bacteria that may serve as protagonists of human CRC. Table 1 summarizes how the data on colon carcinogenesis promoted by these individual bacteria align with criteria supportive of disease causality as classified by Evans AS (1976) and Fredricks DA and Relman DA (1996).

A Consortium as Protagonist

The concept of a keystone species was introduced into environmental science in 1969 and is defined as a species that plays a critical role in maintaining the structure of an ecological community and whose impact on the community is greater than would be expected based on its relative abundance or total biomass (http://www.washington.edu/research/pathbreakers/1969g.html). Consistent with this idea, a number of studies seeking to catalog and categorize the complex microbiota of humans have suggested that minority microbiota members may be prime contributors to microbiota function; in some cases, single species or a limited set of species may serve to distinguish the populations under study (Arumugam et al., 2011; Koren et al., 2013; Qin et al., 2012). Further, the field of microbial communication through quorum sensing and secretion of hormones or anti-bacterial factors such as bacteriocins strongly suggests that individual bacteria possess the capacity to modify, if not commandeer, their nearby microbial community. Given these data and the complexity of the colonic microbiota, the concept of a microbial leader that recruits a consortium of disease-facilitating microbes to initiate the biologic events causing CRC is appealing (Hajishengallis et al., 2012; Sears and Pardoll, 2011; Tjalsma et al., 2012). After all, given the complexity of the colonic microbiota, could all but one microbe be simply passengers not contributing to disease development? Despite its appeal, as yet, no study has specifically tested this idea in the causation of CRC and, thus, this is an area where carefully designed experimental work might help further our concepts of how the microbiota contribute to the pathogenesis of colon carcinogenesis.

Over the decades it takes for human colorectal cancers to develop, several microbial actors may be primary or secondary contributors. Their biomolecular activities in relation to host physiology and in response to a host’s diet or ingested pharmaceuticals may be the factors that explicate environment and human gene interactions in cancer causation and offer up new cancer biomarkers and therapeutic targets. While well-designed microbial discovery efforts are still warranted in human CRC, preclinical models and other approaches which consider the contribution of microbial metabolism to cancer prevention, development, and treatment merit further investigation. Given our evolving understanding of the microbiome, longstanding clinical observations, such as the connections between bile acids or dietary components and gastrointestinal cancers, should be re-examined with a fresh perspective. With this in mind, we will explore select aspects of microbial metabolism in colorectal carcinogenesis and treatment that are more speculative in terms of their mechanistic roles, but represent areas warranting reexamination and further investigation.

High fat diet and CRC: a microbial link?

Diets rich in saturated fats increase bile acid production, and numerous studies have identified associations between diets high in saturated fats and CRC (Williams et al., 2010 and Reddy 2002). Gut bacteria are important contributors to bile acid metabolism and thus may play a role in the biology linking bile acids to CRC. As illustrated in Figure 3, there is significant interplay between host and microbe in bile acid metabolism. The liver secretes glycine and taurine conjugates of two bile acids, cholic and chenodeoxycholic acid. Conjugated bile acids can be deconjugated by bacteria to produce the secondary bile acids, lithocholic and deoxycholic acid, the two dominant fecal bile acids.

Both deoxycholic acid and lithocholic acid may contribute to carcinogenesis. In studies of dietary fat intake and CRC risk, elevated fecal lithocholic and deoxycholic acid have been found in CRC patients relative to healthy controls (Gill and Rowland, 2002). Vancomycin-based perturbations of the gut microbiota support that bacteria are key drivers of high-fat diet-mediated increases in deoxycholic acid in mice (Yoshimoto et al., 2013). Both lithocholic acid and deoxycholic acid function can enhance mutagenesis in classic mutagenesis test, like the Ames test, (Kawalek et al., 1983; Shibuya et al., 1997). In rodent models of colorectal carcinogenesis employing chemical carcinogens, deoxycholic acid and lithocholic acid potentiated tumor number and invasivity, when their endogenous levels were altered by surgery, high-fat diet, or by delivery of supraphysiologic doses to the gastrointestinal tract (Reddy, 1975; Summerton et al., 1985). Focusing on carcinogen-induced liver cancer in mice, Yoshimoto et al. found that a high-fat diet or genetic susceptibility to obesity resulted in deoxycholic acid-mediated induction of the senescence-associated secretory phenotype (secretion of pro-inflammatory and tumor permissive factors by fibroblast-type cells) in hepatic stellate cells and potentiated the development of liver cancer. Notably, perturbing gut bacterial-mediated deoxycholic acid production was sufficient to blunt the development of liver cancer in this model.

Numerous mechanisms underpinning lithocholic and deoxycholic role in carcinogenesis are likely at play. Both lithocholic and deoxycholic acid can be pro-inflammatory in that they can elicit reactive oxygen and nitrogen species production and NF-κB activation in intestinal epithelial cells (Da Silva et al., 2012; Lee et al., 2004; Mühlbauer et al., 2004; Payne et al., 2007). Chronic exposure to deoxycholic acid in vitro results in DNA adducts as well as enhanced epithelial cell proliferation and decreased apoptosis (Barrasa et al., 2013). Thus, lithocholic and deoxycholic acid may represent pro-carcinogenic bacterial metabolites and promising therapeutic targets.

Devkota et al., identified a mechanism that may explain the observations about diets high in saturated fats and escalations in inflammatory diseases in industrialized nations (Devkota et al., 2012). Using Il10^−/− mice, the investigators found that a diet enriched in milk-derived saturated fat versus polyunsaturated-fat altered the composition and function of the gut microbiota. A milk fat-enriched diet enhanced taurine (2-aminoethanesulfonic acid) conjugation of primary bile acids and resulted in a bloom of the sulfur-reducing bacterium Bilophila wadsworthia, which in turn exacerbated colitis in inflammation-prone Il10^−/− mice. A recent human feeding intervention study also supports that a high-fat diet may result in blooms in B. wadsworthia (David et al., 2013). Examining the intersections between dietary fat intake, gut microbiota composition and function, and both primary and secondary bile acids may hold promise for not only mitigating inflammatory disease like colitis but also for attenuating the smoldering inflammation that contributes to sporadic CRC.

Beyond dietary modification, there are other aspects of bile acid microbial metabolism that may offer opportunities for cancer prevention. In contrast to lithocholic and deoxycholic acid, ursodeoxycholic acid has a promising safety and health benefit profile in humans and CRC7 pre-clinical models. Two enzymes, α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase, catalyze the transformation of chenodeoxycholic acid to ursodeoxycholic acid. In vitro many human intestinal bacteria, including Clostridium, Ruminococcus, and Eubacterium strains, can convert chenodeoxycholic acid to ursodeoxycholic acid. However, it is important that a given strain not also express 7β-dehydroxylase, which would convert the ursodeoxycholic acid to the potentially toxic lithocholic acid. Ursodeoxycholic acid is an approved therapy for primary biliary cirrhosis and is generally well-tolerated. A few small, retrospective studies examined if ursodeocycholic acid was useful for CRC prevention in patients with a history of adenomas and inflammatory bowel diseases, however none of these studies were sufficiently powered to demonstrate efficacy (Carey and Lindor, 2012; Serfaty, 2012). Screening, selection, and identification of human intestinal bacteria that can generate ursodeoxycholic acid from chenodeoxycholate in the small intestine could represent a microbe-based approach for optimizing bile acid metabolism for CRC prevention (Lepercq et al., 2004).

Xenobiotic Metabolism, the Gut Microbiota, and Colon Cancer

Many compounds from pharmaceuticals to chemical carcinogens, collectively referred to as xenobiotics, are metabolized in the liver and undergo further metabolism by the gut microbiota (see Figure 3). The liver tends to engage in metabolism via oxidation and conjugation reactions while the gut microbiota favor reduction and hydrolytic processes (Sousa et al., 2008). In the liver some compounds are conjugated to glucuronic acid, which makes them more water-soluble and facilitates their excretion in the urine and feces. Colonic bacterial β-glucuronidases can hydrolyze these conjugates, resulting in their release within the colonic lumen. This joint host and microbe co-metabolism is necessary for the chemical carcinogen azoxymethane to induce intestinal tumors in mice (Fiala, 1977; Takada et al., 1982) and can also contribute to the toxicity of many over-the-counter and prescription drugs (Saitta et al., 2013).

Irinotecan, a commonly used intravenously-delivered CRC chemotherapy, is a topoisomerase-1 inhibitor. Like many chemotherapies, irinotecan can cause both immune suppression and diarrhea. However, in some patients, irinotecan can cause a severe and refractory diarrhea that requires hospitalization and limits the drug’s subsequent dosing and usage. Irinotecan is a prodrug and carboxylesterases, present in the serum and throughout the body, convert it to SN-38. SN-38 is a topoisomerase-1 inhibitor 1000X more potent that irinotecan, and it is glucuronidated in the liver (Kawato et al., 1991). However, within the intestinal lumen, bacterial β-glucuronidase can liberate SN-38 (Roberts et al., 2013). Thus the levels of intestinal bacterial β-glucuronidase and subsequent degree of intestinal epithelial SN-38 exposure influence the drug toxicity for patients. In an elegant study, the Redinbo laboratory identified a potent β-glucuronidase inhibitor that alleviated irinotecan toxicity in mice (Wallace et al., 2010) and in subsequent studies they have identified a number of compounds that are both microbially-selective and have EC50s in the nanomolar range (Roberts et al., 2013). The identification of compounds that can improve drug efficacy and reduce toxicity represents an exciting direction for microbiota-based oncology therapeutics.

The gut microbiota may also contribute to chemotherapy responsiveness via its influence on immune system function. Recently Trinchieri and Goldzmid examined how oxaliplatinum, a drug used to treat CRC, depends on the gut microbiota-immune system interactions for the host to reap full benefits of its anti-cancer effects (Iida et al., 2013 and see review by Trinchieri and Goldzmid in this issue). While chronic exposure to reactive oxygen species can increase the risk of developing CRC, tumor exposure to reactive oxygen species coincident with exposure to DNA adduct-forming platinum-based chemotherapies can be the death knell for cancer cells. Iida et al. found that the gut microbiota influence the expression of several enzymes that myeloid cells require to make reactive oxygen species in the tumor microenvironment. These reactive oxygen species were important for optimal response to specific chemotherapies. In mice, the microbiota’s influence on the immune system appears not only important for homeostatic regulation but also for response to cancer therapy. In a second study, Zitvogel and colleagues identified a gut microbe-dependent mechanism by which cyclophosphamide, a chemotherapy used to treat many non-colorectal cancers, injures the small intestine and triggers anti-tumor Th1 and Th17 immune responses (Viaud et al., 2013).

While the studies of Iida et al. and Viaud et al. are of great interest, there are numerous limitations that need to be considered for the translation of such findings to humans. Anatomical, behavioral and dietary differences, which distinguish mice from humans, influence luminal and mucosal intestinal microbial communities, their transcriptomes, and metabolomes. Thus beyond identifying clades that track with chemotherapy responses, subsequent studies need to address how microbial pattern recognition receptor and metabolite signaling in the host, specifically human hosts, can be tuned to both mitigate side effects and optimize tumor responsiveness to chemotherapies. This point requires especial consideration as cancer patients often require antibiotics for bacterial infections while receiving chemotherapy. An over-arching message of the above studies is that we need to develop a well-nuanced understanding of how drug and biologic treatments alter the gut microbiota and immune system function.

Conclusion

Cancer has been called the ‘emperor of all maladies’ (Mukherjee 2010) and in unraveling the role of the microbiota in colorectal carcinogenesis, research efforts are giving this emperor new clothes and laying him bare. With sufficient research support the vast genomic and metabolic potential of the gut microbiota may be realized as the most powerful weapon in the 40-plus year war on cancer. Specific species, microbial consortia, and microbial metabolites generated from ingested foodstuffs are all potential targets for decreasing or increasing cancer risk and perhaps even for diagnosis, treatment stratification, and therapy.