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...

Mutations in the adenomatous polyposis coli or ␤-catenin gene lead to cytosolic accumulation of ␤-catenin and, subsequently, to increased transcriptional activity of the ␤-catenin-T cell-factor/lymphoid-enhancer-factor complex. This process seems to play an essential role in the development of most colorectal carcinomas. To identify genes activated by ␤-catenin overexpression, we used colorectal cell lines for transfection with the ␤-catenin gene and searched for genes differentially expressed in the transfectants. There are four genes affected by ␤-catenin overexpression; three overexpressed genes code for two components of the AP-1 transcription complex, c-jun and fra-1, and for the urokinase-type plasminogen activator receptor (uPAR), whose transcription is activated by AP-1. The direct interaction of the ␤-catenin-T cell-factor͞lymphoid-enhancerfactor complex with the promoter region of c-jun and fra-1 was shown in a gel shift assay. The concomitant increase in ␤-catenin expression and the amount of uPAR was confirmed in primary colon carcinomas and their liver metastases at both the mRNA and the protein levels. High expression of ␤-catenin in transfectants, as well as in additionally analyzed colorectal cell lines, was associated with decreased expression of ZO-1, which is involved in epithelial polarization. Thus, accumulation of ␤-catenin indirectly affects the expression of uPAR in vitro and in vivo. Together with the other alterations, ␤-catenin accumulation may contribute to the development and progression of colon carcinoma both by dedifferentiation and through proteolytic activity.

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Our results suggest that CCAL is a crucial oncogenic regulator involved in CRC tumorigenesis and progression.