Published online Dec 21, 2018. doi: 10.3748/wjg.v24.i47.5351
Peer-review started: October 30, 2018
First decision: November 6, 2018
Revised: November 29, 2018
Accepted: December 13, 2018
Article in press: December 13, 2018
Published online: December 21, 2018
Recently, Fusobacterium nucleatum (F. nucleatum), an anaerobic bacterial component of the oral and gut commensal flora, has emerged as a risk factor for colorectal cancer (CRC) development. Several studies have observed an association between overabundance of F. nucleatum in colonic tumor tissue compared to the normal matched mucosa. However, despite progress in this field the molecular mechanisms of how the bacterium etiologically contributes to carcinogenesis are still unclear.
We previously observed an association of the TLR2-196 to -174del genetic variant with increased CRC risk, together with an increased expression of TLR2 mRNA and protein in tumor tissues[31]. The major postulated mechanism of F. nucleatum-mediated colorectal tumorigenesis involves immune related inflammatory responses. Therefore, we decided to extend our previous work by measuring the transcript levels of important mediators in the pathogen-activated immune and inflammatory response, including TLR2 / TLR4 receptor and cytokine genes, and then evaluating the association of their expression with F. nucleatum levels in colorectal tumors. As microRNAs have been shown to be epigenetic regulators of inflammatory responses, we further examined the involvement of miRNAs in modulating the bacterial - cytokine interaction.
The main objective of this study was to investigate the association between inflammatory genes and F. nucleatum in colorectal carcinogenesis, by examining tissues from the major colorectal neoplasms of adenoma and adenocarcinomas. A secondary objective was to examine the interaction of the bacterial mediated immune response with microRNA (miRNA) regulation. The elucidation of likely mechanisms whereby F. nucleatum may contribute to inflammatory mediated colorectal carcinogenesis will help to better understand the molecular pathways activated by this bacterium and where prevention and treatment strategies can be best targeted.
Robust techniques were used for DNA quantification of F. nucleatum and RNA transcript measures of the inflammatory genes and miRNAs in normal and tumor tissues. For this purpose, we used TaqMan gene expression assays (Applied Biosystems, Foster City, CA, United States) with specific probes for each gene and miRNA for relative quantification. The reactions were analyzed using the StepOnePlus real-time PCR System (Applied Biosystems, Foster City, CA, United States). Mutation testing of the KRAS gene was performed by direct sequencing and microsatellite instability (MSI) evaluation was performed using a multiplex PCR. In addition, we also used a bioinformatic tool ‘miRNA Data Integration Portal’ (http://ophid.utoronto.ca/mirDIP/)[34] to build an miRNA:mRNA interaction network by using Cytoscape software (version 3.1.1)[36].
Ours results confirm the overabundance of F. nucleatum in adenoma and tumor neoplasms compared to their respective matched normal tissues, as previously found in several populations. We further suggest that this bacterial load increases the expression of TLR2 and TLR4 receptors and consequently of pro-inflammatory interleukins IL1B, IL6 and IL8. This immune-modulation of the inflammatory response to F. nucleatum colonic invasion also affects the expression of miRNA regulators of the inflammatory response. In particular, these miRNA:mRNA interactions network indicate a mechanism of colorectal carcinogenesis where altered expression of miR-34a, miR-135b, and miR-22, previously associated with CRC, occurs via a TLR2/TLR4 dependent response to F. nucleatum. In analyses stratified by tumor molecular characteristics, we observed that KRAS was more frequently mutated in tumors with F. nucleatum, and that an increased IL-8 expression was associated with MSI-high status. Therefore, more studies of gene function and regulation within the inflammatory pathways impacted by F. nucleatum invasion are needed, along with consideration of tumor molecular subtypes.
Our findings reinforce the increasing invasion of F. nucleatum during the colorectal adenoma to cancer development. This appears to increase expression of pro-inflammatory mediators and dysregulation of miRNA expression, leading to a more carcinogenic microenvironment alongside genetic alterations such as KRAS mutation and MSI-high. Therefore, together with other studies, our results suggest that F. nucleatum is involved in CRC development through immune responses to inflammatory stresses. Further work is needed to functionally demonstrate these postulated tumorigenic mechanisms, and also for early CRC detection and diagnosis strategies using biomarkers of F. nucleatum presence or the consequent immune response.
The intestinal microbiota is very diverse and important for the maintenance of epitelial homeostasis. Disturbances of this microbiome balance appears to be a major factor in CRC etiology. F. nucleatum has been implicated in recent years, by in vitro and in mouse models, as a carcinogenic bacterium through generation of a microenvironment conducive to cancer development. Considering that F. nucleatum has been found to be highly abundant in both adenoma and CRC neoplasms, it may have uses as a tissue or non-invasive biomarker in faeces (or possibly mouth-rinse samples) for CRC and the early detection of adenomas (which may help define a higher risk group for CRC development due to the presences of the bacterium). However, further investigations are needed to understand the molecular mechanisms in the immuno-inflammatory response to the increased invasion of this bacterium into developing neoplasms, and if this can promote genetic and epigenetic alterations that may culminate in CRC development.