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

Abstract

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 buffer 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 (si) 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 APCmin/+, C57BL miR21a−/−, and C57BL mice with full-length miR21a (controls) were given F nucleatum by gavage; some mice were given azoxymethane (AOM) and dextran sodium sulfate (DSS) to induce colitis and colon tumors. Intestinal tissues were collected and tumors were counted. Serum samples from mice were analyzed for cytokine levels by ELISAs. We performed in situ hybridization analyses to detect enrichment of F nucleatum in CRC cells. F nucleatum DNA in 90 tumor and matched non-tumor 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 F 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. APCmin/+ 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 [IL17F], IL21, IL22, and MIP3A). We found 50 miRNAs to be significantly upregulated and 52 miRNAs to be significantly downregulated in CRCs incubated with F nucleatum vs PBS; levels of miR21 increased by the greatest amount (more than 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 AOM and DSS, 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 a RAS GTPase, to be one of the target genes consistently downregulated in cells that overexpressed miR21 and upregulated in cells exposed to miR21 inhibitors. Infection of cells with F nucleatum increased expression of miR21 by activating TLR4 signaling to MYD88, leadi...

Relationship of the mucosal microbiota to gastrointestinal inflammation and small cell intestinal lymphoma in cats

“…Fusobacterium nucleatum numbers are significantly correlated with tumor size, and shortened survival times in a population of human Japanese patients with later stage CRC compared to earlier stages 53. These findings, as well as additional observations in rodent models and humans with CRC suggest that chronic inflammatory disease of the GI tract is a potential risk factor for the development of additional channels for amplified inflammatory processes, aneuploidy, high‐grade dysplasia, metaplasia and loss of cellular proliferative checkpoints, all of which are processes that can be appreciated in malignancy 13, 19, 54, 58, 59. The increased numbers of mucosal Fusobacterium spp.…”

“…Furthermore, CD11b + myeloid cells might contribute to development of a tumor microenvironment,19, 63 and Fusobacterium spp. might induce toll like receptors (TLR), to stimulate NF‐κB through the TLR4/MYD88 pathway 54. Importantly, TLRs are found on both immune and non‐immune cells, and can be expressed on tumors including human mantle cell lymphoma 21, 64…”

“…This is worth assessing in future studies, as in human studies and rodent models, overabundance of Fusobacterium spp. in patients or rodents with CRC does appear to have a correlation with poorer prognostic outcome 36, 51, 54…”

<i>Fusobacterium nucleatum</i> Infection in Colorectal Cancer: Linking Inflammation, DNA Mismatch Repair and Genetic and Epigenetic Alterations

“…Similar to Kostic’s results, introduction of Fn increased the number of tumors in C57BL/6 Apc Min/+ mice (18). Similarly, Yang et al showed that introduction of Fn into C57BL/6 Apc Min/+ mice increased the number and size of tumors, and shortened overall survival compared with the non-treated controls (19). Although these three studies suggest that Fn infection may promote loss of a wild-type copy of Apc in Apc Min/+ colon cells to form adenoma, they do not give evidence that Fn infection may be involved in adenoma-to-carcinoma transition in the colon and rectum.…”

“…Increased loads of Fn are associated with advanced CRC stages (6,16–18) and with poor prognoses (19,43–46). Mima et al showed that colon cancer-specific death, but not overall survival is associated with high levels of tissue Fn (43).…”