Fusobacterium nucleatum Increases Proliferation of Colorectal Cancer Cells and Tumor Development in Mice by Activating Toll-Like Receptor 4 Signaling to Nuclear Factor-κB, and Up-regulating Expression of MicroRNA-21.

BACKGROUND & AIMS: Nearly 20% of the global cancer burden can be linked to infectious agents. Fusobacterium nucleatum promotes tumor formation by epithelial cells via unclear mechanisms. We aimed to identify microRNAs (miRNAs) induced by F nucleatum and evaluate their ability to promote colorectal carcinogenesis in mice. METHODS: Colorectal cancer (CRC) cell lines were incubated with F nucleatum or control reagents and analyzed in proliferation and would healing assays. HCT116, HT29, LoVo, and SW480 CRC cell lines were incubated with F nucleatum or phosphate-buffered saline (PBS [control]) and analyzed for miRNA expression patterns and in chromatin immunoprecipitation assays. Cells were incubated with miRNAs mimics, control sequences, or small interfering RNAs; expression of reporter constructs was measured in luciferase assays. CRC cells were incubated with F nucleatum or PBS and injected into BALB/C nude mice; growth of xenograft tumors was measured. C57BL adenomatous polyposis colimin/+, C57BL miR21a-/-, and C57BL mice with full-length miR21a (controls) were given F nucleatum by gavage; some mice were given azoxymethane and dextran sodium sulfate to induce colitis and colon tumors. Intestinal tissues were collected and tumors were counted. Serum samples from mice were analyzed for cytokine levels by enzyme-linked immunosorbent assay. We performed in situ hybridization analyses to detect enrichment of F nucleatum in CRC cells. Fusobacterium nucleatum DNA in 90 tumor and matched nontumor tissues from patients in China were explored for the expression correlation analysis; levels in 125 tumor tissues from patients in Japan were compared with their survival times. RESULTS: Fusobacterium nucleatum increased proliferation and invasive activities of CRC cell lines compared with control cells. CRC cell lines infected with F nucleatum formed larger tumors, more rapidly, in nude mice than uninfected cells. Adenomatous polyposis colimin/+ mice gavaged with F nucleatum developed significantly more colorectal tumors than mice given PBS and had shorter survival times. We found several inflammatory factors to be significantly increased in serum from mice given F nucleatum (interleukin 17F, interleukin 21, and interleukin 22, and MIP3A). We found 50 miRNAs to be significantly up-regulated and 52 miRNAs to be significantly down-regulated in CRCs incubated with F nucleatum vs PBS; levels of miR21 increased by the greatest amount (>4-fold). Inhibitors of miR21 prevented F nucleatum from inducing cell proliferation and invasion in culture. miR21a-/- mice had a later appearance of fecal blood and diarrhea after administration of azoxymethane and dextran sodium sulfate, and had longer survival times compared with control mice. The colorectum of miR21a-/- mice had fewer tumors, of smaller size, and the miR21a-/- mice survived longer than control mice. We found RASA1, which encodes an RAS GTPase, to be one of the target genes consistently down-regulated in cells that overexpressed miR21 and up-regulated in cells exposed to miR21 inhibitors. Infection of cells with F nucleatum increased expression of miR21 by activating Toll-like receptor 4 signaling to MYD88, leading to activation of the nuclear factor-κB. Levels of F nucleatum DNA and miR21 were increased in tumor tissues (and even more so in advanced tumor tissues) compared with non-tumor colon tissues from patients. Patients whose tumors had high amounts of F nucleatum DNA and miR21 had shorter survival times than patients whose tumors had lower amounts. CONCLUSIONS: We found infection of CRC cells with F nucleatum to increase their proliferation, invasive activity, and ability to form xenograft tumors in mice. Fusobacterium nucleatum activates Toll-like receptor 4 signaling to MYD88, leading to activation of the nuclear factor-κB and increased expression of miR21; this miRNA reduces levels of the RAS GTPase RASA1. Patients with both high amount of tissue F nucleatum DNA and miR21 demonstrated a higher risk for poor outcomes.

Association analysis of dietary habits with gut microbiota of a native Chinese community.

Environmental exposure, including a high-fat diet (HFD), contributes to the high prevalence of colorectal cancer by changing the composition of the intestinal microbiota. However, data examining the interaction between dietary habits and intestinal microbiota of the Chinese population is sparse. We assessed dietary habits using a food frequency questionnaire (FFQ) in native Chinese community volunteers. Based on the dietary fat content determined using the FFQ, the volunteers were divided into HFD group (≥40% of dietary calories came from fat) or low-fat diet (LFD) group (<40%). Fecal and colonic mucosal microbiota composition was determined using 16S rDNA based methods. In stool matter of HFD group, Prevotella and Abiotrophia showed significantly higher abundance, whereas unclassified genus of S24-7 (family level) of Bacteroidetes, Gemmiger, Akkermansia and Rothia were less abundant. On colonic mucosal tissue testing, unclassified genus of S24-7 showed significantly higher abundance while Bacteroides, Coprobacter, Abiotrophia, and Asteroleplasma were less abundant in HFD group. A high fat and low fiber diet in a native Chinese community may partially contribute to changes of intestinal microbiota composition that may potentially favor the onset and progression of gastrointestinal disorders including inflammatory, hyperplastic and neoplastic diseases.

Systematic literature review and clinical validation of circulating microRNAs as diagnostic biomarkers for colorectal cancer.

Because patients with colorectal cancer (CRC) are usually diagnosed at an advanced stage and current serum tumor markers have limited diagnostic efficacy, there is an urgent need to identify reliable diagnostic biomarkers. To better define the diagnostic potential of microRNAs (miRNAs) for CRC, we performed a comprehensive evaluation of reported circulating CRC miRNA markers. After a systematic literature review, we selected 30 candidate miRNAs and used quantitative real-time polymerase chain reaction to examine their expression in a training cohort of 120 plasma samples (CRC vs healthy controls (HC) = 60:60). Expression data was confirmed in a validation cohort of 160 plasma samples (CRC vs HC = 80:80). We ultimately identified 5 dysregulated circulating miRNAs (miR-15b, miR-17, miR-21, miR-26b, and miR-145), of which miR-21 and miR-26b proved to have the best diagnostic performance in the training and validation cohorts, respectively. Based on these results, we propose a novel blood-based diagnostic model, integrating 5 CRC-related miRNAs and serum carcinoembryonic antigen (CEA), which provides better diagnostic performance than the combined 5 miRNAs, CEA alone, or any single miRNA. We propose that the novel CRC diagnostic model presented here will be useful for overcoming the limitations faced by current non-invasive diagnostic strategies.

The effect of perioperative probiotics treatment for colorectal cancer: short-term outcomes of a randomized controlled trial.

This study was designed to mainly evaluate the anti-infective effects of perioperative probiotic treatment in patients receiving confined colorectal cancer (CRC) respective surgery. From November 2011 to September 2012, a total of 60 patients diagnosed with CRC were randomly assigned to receive probiotic (n = 30) or placebo (n = 30) treatment. The operative and post-operative clinical results including intestinal cleanliness, days to first - flatus, defecation, fluid diet, solid diet, duration of pyrexia, average heart rate, length of intraperitoneal drainage, length of antibiotic therapy, blood index changes, rate of infectious and non-infectious complications, postoperative hospital stay, and mortality were investigated. The patient demographics were not significantly different (p > 0.05) between the probiotic treated and the placebo groups. The days to first flatus (3.63 versus 3.27, p = 0.0274) and the days to first defecation (4.53 versus 3.87, p = 0.0268) were significantly improved in the probiotic treated patients. The incidence of diarrhea was significantly lower (p = 0.0352) in probiotics group (26.67%, 8/30) compared to the placebo group (53.33%, 16/30). There were no statistical differences (p > 0.05) in other infectious and non-infectious complication rates including wound infection, pneumonia, urinary tract infection, anastomotic leakage, and abdominal distension. In conclusion, for those patients undergoing confined CRC resection, perioperative probiotic administration significantly influenced the recovery of bowel function, and such improvement may be of important clinical significance in reducing the short-term infectious complications such as bacteremia.