title: 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: 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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Department of Colorectal Surgery, Fudan University Shanghai Cancer Center, Shanghai, China; Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China; Department of Gastrointestinal Surgery, Shanghai Tenth People's Hospital Affiliated to Tongji University, Shanghai, China.
authors.forename: Yongzhi
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Center for Gastrointestinal Research, Center for Translational Genomics and Oncology, Baylor Scott & White Research Institute and Charles A. Sammons Cancer Center, Dallas, Texas; Department of Clinical Laboratory, Shanghai Tenth People's Hospital Affiliated to Tongji University, Shanghai, China.
authors.forename: Wenhao
authors.initials: W
authors.lastname: Weng
authors.affiliation: Department of Colorectal Surgery, Fudan University Shanghai Cancer Center, Shanghai, China; Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China.
authors.forename: Junjie
authors.initials: J
authors.lastname: Peng
authors.affiliation: Department of Gastrointestinal Surgery, Shanghai Tenth People's Hospital Affiliated to Tongji University, Shanghai, China.
authors.forename: Leiming
authors.initials: L
authors.lastname: Hong
authors.affiliation: Department of Gastrointestinal Surgery, Shanghai Tenth People's Hospital Affiliated to Tongji University, Shanghai, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Yang
authors.affiliation: Division of Reparative Medicine, Department of Gastrointestinal and Pediatric Surgery, Institute of Life Sciences, Mie University Graduate School of Medicine, Mie, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Toiyama
authors.affiliation: Department of Gastrointestinal Surgery, Shanghai Tenth People's Hospital Affiliated to Tongji University, Shanghai, China.
authors.forename: Renyuan
authors.initials: R
authors.lastname: Gao
authors.affiliation: Department of Gastrointestinal Surgery, Shanghai Tenth People's Hospital Affiliated to Tongji University, Shanghai, China.
authors.forename: Minfeng
authors.initials: M
authors.lastname: Liu
authors.affiliation: Department of Gastrointestinal Surgery, Shanghai Tenth People's Hospital Affiliated to Tongji University, Shanghai, China.
authors.forename: Mingming
authors.initials: M
authors.lastname: Yin
authors.affiliation: Department of Gastrointestinal Surgery, Shanghai Tenth People's Hospital Affiliated to Tongji University, Shanghai, China.
authors.forename: Cheng
authors.initials: C
authors.lastname: Pan
authors.affiliation: Department of Gastrointestinal Surgery, Shanghai Tenth People's Hospital Affiliated to Tongji University, Shanghai, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Li
authors.affiliation: Department of Surgery, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China.
authors.forename: Bomin
authors.initials: B
authors.lastname: Guo
authors.affiliation: Department of Surgery, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China.
authors.forename: Qingchao
authors.initials: Q
authors.lastname: Zhu
authors.affiliation: Department of Pathology, Shanghai Tenth People's Hospital Affiliated to Tongji University, Shanghai, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Wei
authors.affiliation: INCELL Corporation, San Antonio, Texas.
authors.forename: Mary-Pat
authors.initials: MP
authors.lastname: Moyer
authors.affiliation: Department of Central Laboratory, Shanghai Tenth People's Hospital of Tongji University, School of Life Science and Technology, Tongji University, Shanghai, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Wang
authors.affiliation: Department of Colorectal Surgery, Fudan University Shanghai Cancer Center, Shanghai, China; Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China. Electronic address: sanjuncai@hotmail.com.
authors.forename: Sanjun
authors.initials: S
authors.lastname: Cai
authors.affiliation: Center for Gastrointestinal Research, Center for Translational Genomics and Oncology, Baylor Scott & White Research Institute and Charles A. Sammons Cancer Center, Dallas, Texas. Electronic address: Ajay.Goel@BSWHealth.org.
authors.forename: Ajay
authors.initials: A
authors.lastname: Goel
authors.affiliation: Department of Gastrointestinal Surgery, Shanghai Tenth People's Hospital Affiliated to Tongji University, Shanghai, China. Electronic address: hl-qin@hotmail.com.
authors.forename: Huanlong
authors.initials: H
authors.lastname: Qin
authors.affiliation: Department of Colorectal Surgery, Fudan University Shanghai Cancer Center, Shanghai, China; Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China. Electronic address: yanleima@live.cn.
authors.forename: Yanlei
authors.initials: Y
authors.lastname: Ma
mesh_terms: D025601:Adenomatous Polyposis Coli Protein / Q000235:genetics; D000368:Aged; D000818:Animals; D001397:Azoxymethane; D063646:Carcinogenesis; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D003092:Colitis / Q000139:chemically induced; D003110:Colonic Neoplasms / Q000737:chemistry / Q000235:genetics / Q000382:microbiology* / Q000473:pathology; D004269:DNA, Bacterial / Q000032:analysis*; D016264:Dextran Sulfate; D015536:Down-Regulation; D005260:Female; D005674:Fusobacterium Infections / Q000235:genetics*; D016967:Fusobacterium nucleatum; D015972:Gene Expression Regulation, Neoplastic; D045325:HCT116 Cells; D019073:HT29 Cells; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D035683:MicroRNAs / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D053594:Myeloid Differentiation Factor 88 / Q000378:metabolism; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011379:Prognosis; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation; D020729:p120 GTPase Activating Protein / Q000235:genetics
chemical_list: D025601:Adenomatous Polyposis Coli Protein; D001397:Azoxymethane; D004269:DNA, Bacterial; D016264:Dextran Sulfate; D035683:MicroRNAs; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B; D034741:RNA, Small Interfering; D051197:Toll-Like Receptor 4; D020729:p120 GTPase Activating Protein; C524940:MIRN21 microRNA, human; C557248:RASA1 protein, human; C581321:RASA1 protein, mouse; C531950:MIRN21 microRNA, mouse
keywords: Carcinogenesis; Gene Regulation; Microbe; Signal Transduction