A collaborative team of researchers in France has demonstrated that an imbalance in gut microbiota, or dysbiosis, promotes the onset of colorectal cancer (CRC). The scientists, who have worked together as the Oncomix group since April 2016, showed that transplanting fecal microbiota from patients with colon cancer into mice caused lesions and epigenetic changes that were characteristic of the development of a malignant tumor. The studies led to the development of a noninvasive blood test that can identify the epigenetic features associated with dysbiosis. The blood test was validated in 1,000 individuals.
The research groups included the gastroenterology team at Henri-Mondor AP-HP Hospital and University Paris-Est Créteil, in France, led by Iradj Sobhani, Ph.D., together with teams from Inserm and the Institut Pasteur Molecular Microbial Pathogenesis Unit (U1202), led by Philippe Sansonetti Ph.D., who holds the microbiology and infectious diseases chair at the Collège de France. Their research is reported in the Proceedings of the National Academy of Sciences (PNAS), in a paper titled, “Colorectal cancer-associated microbiota contributes to oncogenic epigenetic signatures.”
Sporadic colorectal cancer can develop in patients in the absence of any known risk factors, and is the result of “complex interactions between the host and its environment,” the authors wrote. CRC is among the most common form of cancer worldwide, and incidence of the disease is increasing, which may reflect our increased exposure to environmental insults that might cause an accumulation of cancer-promoting DNA mutations, or epigenetic changes to DNA within host cells. However, the authors stated, comprehensively assessing every potential contributory factor is an “extremely challenging endeavor, primarily due to the great variety of environmental stressors and the long-term exposure period of many years.”
Several studies have investigated the role of the microbiota as a mediator of these interactions. “… significant associations linking diet to features of the microbiota and to CRC were reported recently,” they wrote. “Interestingly, it has been reported that microbiota dysbiosis increases the risk for CRC even in individuals with genetic predisposition to CRC, including constitutional mutations of pivotal genes … Therefore, analyzing the microbiota may provide new insights into the effects of environmental exposures on CRC.”
For their reported research, the investigators first characterized the microbiota in human colorectal cancer patients, and then evaluated the effects of transferring fecal samples from nine patients with sporadic colorectal cancer patients into germ-free mice that have no natural gut microbiome. A separate cohort of mice received fecal transplants from healthy donors. The animals’ colons were examined 7 and 14 weeks after they received the human fecal microbiota transplant. The investigators were particularly interested in assessing the number and development of aberrant crypt foci (or ACF, a type of precancerous lesion), analyzing the resulting microbial profile in the animal recipients, and evaluating any damage to colonic DNA. They also took into account the animals’ food intake, weight, and blood indicators.
A link between fecal dysbiosis and the genetic and epigenetic DNA signatures in the animals’ tissues was identified using statistical tests. The results suggested that mice which had received a fecal microbiota transfer (FMT) from patients with sporadic colorectal cancer developed precancerous lesions known as aberrant crypt foci (ACF), without any significant genetic changes to the colon, but they also had a greater number of hypermethylated genes, which have been significantly linked to the incidence of ACF in the colonic mucosa. The results indicated that “the human CRC FMT may induce systemic changes in mice mediated by the interaction of bacteria with the colonic mucosa,” the team wrote. “Pooling of total genes with expected DNA alterations (i.e., hypermethylation, or mutation in exon) revealed the involvement of pathways implicated in cell growth, signal transduction, nucleic acid binding, protein synthesis, channel, and carrier protein.”
Having verified an association between fecal dysbiosis and DNA methylation changes in the human sporadic CRC patients who took part in the fecal transplant experiments, the investigators also carried out a pilot study in humans with the aim of developing a simple, reproducible blood test that could be used for early-stage diagnosis of colorectal tumors in asymptomatic patients. “To investigate whether the gene methylation patterns observed in the mouse were also associated with CRC dysbiosis in humans, we first investigated methylated genes in CRC tissues and effluents such as blood and stool (n = 9) and in normal tissue effluent controls (n = 9), both obtained from the same individuals as for the FMT in mice. In order to develop a quick and easy methylation test, panels of genes were selected, based on the difference between normal and CRC in various effluents and tissues.”
The researchers found that eight genes were hypermethylated in all the samples evaluated, and they selected three as the representative cumulative methylation index (CMI). The patients were then classified according to their CMI (positive or negative). An analysis identified a positive CMI as a predictive factor for the onset of sporadic colorectal cancer. Having developed the prototype classification, prospective validation of the test was performed on 1,000 asymptomatic patients who were due to be given a colonoscopy.
To identify the bacteria that might be involved in triggering the methylation changes, the entire bacterial genome in fecal samples was sequenced, using 16S rRNA gene sequencing technology. “Interestingly, sequencing of fecal microbiota from CRC patients and normal controls showed that CRC-associated dysbiosis was significantly associated with hypermethylation of several gene promoters.” They found that more than 50% of the CRC patients with symptoms, and 35% of the asymptomatic individuals who had early CRC in the validation study showed a positive CMI, compared with only 4% of individuals with normal colonoscopy.
Further studies showed that compared with healthy controls, the microbiota of CRC patients contained either high or lower proportions of a number of bacteria, and that in the mice this imbalance might be responsible for changes to the colonic mucosa. “… our data present evidence for the association between a dysbiosis and CRC causing alterations in gene methylation,” the authors concluded. “The cohort in which dysbiosis and gene methylation were investigated is the largest to date and strongly indicates a dysbiosis-induced imbalance in gene methylation and in bacterial species. We could identify bacteria species who were significantly associated with higher levels of methylation test (CMI >2) by using whole metagenomic analysis. These measurements can now be proposed as markers for the effectiveness of and adherence to prebiotic and probiotic therapies.”