Published OnlineFirst May 8, 2017; DOI: 10.1158/1940-6207.CAPR-16-0178 Research Article Cancer Prevention Research Fusobacterium Nucleatum Subspecies Animalis Influences Proinflammatory Cytokine Expression and Monocyte Activation in Human Colorectal Tumors Xiangcang Ye1, Rui Wang1, Rajat Bhattacharya1, Delphine R. Boulbes1, Fan Fan1, Ling Xia1, Harish Adoni1, Nadim J. Ajami2, Matthew C. Wong2, Daniel P. Smith2, Joseph F. Petrosino2, Susan Venable3, Wei Qiao4, Veera Baladandayuthapani4, Dipen Maru5, and Lee M. Ellis1,6 Abstract Chronic infection and associated inflammation have using immunoassays and found that expression of the long been suspected to promote human carcinogenesis. cytokines IL17A and TNFa was markedly increased but Recently, certain gut bacteria, including some in the Fuso- IL21 decreased in the colorectal tumors. Furthermore, the bacterium genus, have been implicated in playing a role in chemokine (C-C motif) ligand 20 was differentially human colorectal cancer development. However, the Fuso- expressed in colorectal tumors at all stages. In in vitro co- bacterium species and subspecies involved and their onco- culture assays, F. nucleatum ssp. animalis induced CCL20 genic mechanisms remain to be determined. We sought to protein expression in colorectal cancer cells and monocytes. identify the specific Fusobacterium spp. and ssp. in clinical It also stimulated the monocyte/macrophage activation and colorectal cancer specimens by targeted sequencing of migration. Our observations suggested that infection with Fusobacterium 16S ribosomal RNA gene. Five Fusobacterium F. nucleatum ssp. animalis in colorectal tissue could induce spp. were identified in clinical colorectal cancer specimens. inflammatory response and promote colorectal cancer Additional analyses confirmed that Fusobacterium nuclea- development. Further studies are warranted to determine tum ssp. animalis was the most prevalent F. nucleatum if F. nucleatum ssp. animalis could be a novel target for subspecies in human colorectal cancers. We also assessed colorectal cancer prevention and treatment. Cancer Prev Res; inflammatory cytokines in colorectal cancer specimens 10(7); 398–409. Ó2017 AACR. Introduction 1Department of Surgical Oncology, The University of Texas MD Ander- son Cancer Center, Houston, Texas. 2Alkek Center for Metagenomics The gastrointestinal tract has a high density of commensal and Microbiome Research, Department of Molecular Virology and microbes that are not only important to digestive physiol- Microbiology, Baylor College of Medicine, Houston, Texas. 3Texas ogy but also critical to immune system development and Children's Microbiome Center, Department of Pathology and Immu- function. However, an altered intestinal microbiota is 4 nology, Baylor College of Medicine, Houston, Texas. Department of believed to be a potential risk factor for colorectal carcino- Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, Texas. 5Department of Pathology, The University of Texas genesis (1, 2). Early studies linked certain enteric bacterial fl MD Anderson Cancer Center, Houston, Texas. 6Department of Molec- infections with in ammatory bowel disease and colorectal ular and Cellular Oncology, The University of Texas MD Anderson cancer, particularly pathogenic bacterial species such as Cancer Center, Houston, Texas. Streptococcus gallolyticus (3), Enterococcus faecalis (4), adherent Note: Supplementary data for this article are available at Cancer Escherichia coli (5), and enterotoxigenic Bacteroides fragilis Prevention Research Online (http://cancerprevres.aacrjournals.org/). (6). However, the role of these species as causal factors for Corresponding Authors: L.M. Ellis, The University of Texas MD colorectal carcinogenesis remains to be determined. Anderson Cancer Center, 1400 Pressler St., Unit 1484, Houston, Recent discoveries using next-generation sequencing and TX 77030. Phone: 713-792-6926; Fax: 713-792-4689; E-mail: functional studies established an association between Fuso- [email protected]; and Xiangcang Ye, [email protected] bacterium spp. infection and colorectal cancer development (7, 8). Fusobacterium is a genus of anaerobic Gram-negative doi: 10.1158/1940-6207.CAPR-16-0178 bacteria previously known to be oral pathogens. They Ó2017 American Association for Cancer Research. are mainly associated with dental plaque biofilms and 398 Cancer Prev Res; 10(7) July 2017 Downloaded from cancerpreventionresearch.aacrjournals.org on October 1, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst May 8, 2017; DOI: 10.1158/1940-6207.CAPR-16-0178 Fusobacterium Promotes Inflammation in Colorectal Tumors inflammatory periodontal diseases but are also found in expression are important factors associated with human individuals with extraoral infections (9). Fusobacterium colorectal cancer progression. encompasses at least 15 species (10, 11), of which Fuso- bacterium nucleatum was one of the most frequently iden- Materials and Methods fi ti ed species in periodontal and bowel diseases (9, 12). Cell lines and bacterial strains fi Moreover, the taxonomy of F. nucleatum consists of ve The human colon epithelial cell line CCD841CoN, — subspecies nucleatum, polymorphum, fusiforme, animalis, colorectal cancer cell lines (SW480, HT29, HCT116, and — and vincentii with fusiforme and vincentii indicated to be RKO), and monocyte THP-1 were obtained from the the same subspecies according to genomic analysis (13, ATCC. The human primary colorectal cancer cell line HCP1 14). However, the etiological relationship between the F. was generated in our laboratory under a protocol approved nucleatum subspecies and colorectal cancer development by the Institutional Review Board (IRB) of The University remains to be determined. of Texas MD Anderson Cancer Center (UTMDACC; ref. 29). It is known that colorectal mucosal immunity plays an Cell lines were routinely cultured in minimum essential important role in maintenance of mucosal symbiosis with medium or RPMI 1640 complete medium containing 10% the gut microbiota (15). Chronic relapse and remission of FBS under standard human cell culture conditions at 37C. fl in ammation can result in repeated epithelial injury and The normoxic culture was performed in an incubator with DNA damage, leading to colorectal carcinogenesis (15). 5% CO and 21% O , whereas the hypoxic culture was fl fl 2 2 In amed mucosa and neoplasia secrete proin ammatory carried in 5% CO and 2% O . Cells were free of myco- b 2 2 cytokines, including IL17A, IL1B (IL1 ), IL6, and TNF, all plasma as confirmed using a MycoAlert mycoplasma detec- of which are implicated to promote colorectal tumori- tion kit (Lonza Group). genesis (16, 17). The recruitment of myeloid-derived cells The F. nucleatum ssp. animalis Gharbia and Shah (ATCC fl to the in amed mucosa is partly mediated by chemokine 51191) type strain NCTC 12276 was obtained from the signaling. Chemokine (C-C motif) ligand 20 (CCL20), ATCC. The strain was cultured in CDC anaerobe 5% sheep fl a also known as macrophage in ammatory protein-3 or blood agar plates (Becton Dickinson and Company) under liver activation-regulated chemokine, is one of upregulat- anaerobic conditions using an AnaeroGen Compact system fl ed chemokines in the in ammatory microenvironment (Oxoid) with a 37C incubator. (18, 19). CCL20 protein expression can be stimulated by lipopolysaccharide (LPS) and TNF via the NF-kB pathway Human tissue specimens (20). It is the sole high-affinity ligand for chemokine (C-C Surgical residual colorectal adenoma or adenocarcino- motif) receptor 6 (CCR6; ref. 21). The CCL20/CCR6 axis þ ma and adjacent normal tissue specimens were collected regulates recruitment of CCR6 immune cells, including from patients by members of the Department of Pathology subsets of IL17-expressing T helper cells (Th17), regulatory at UTMDACC. Informed consent for use of the paired T cells, and dendritic cells, to neoplastic lesions (22–25). frozen specimens was obtained from the patients accord- Interestingly, colorectal cancer can hijack CCL20/CCR6 ing to an IRB-approved protocol. Clinical characteristics of function to promote hepatic metastasis of colorectal can- the patients in this study are listed in Supplementary Table cers (19, 26–28). However, the mechanisms involving the S1. Aliquots of each of the tissue specimens were utilized tripartite relationships among specific bacteria, tumor for protein extraction and cytokine panel analysis; the cells, and immune cells in the tumor microenvironments tissue aliquots were also used in DNA extraction and have yet to be fully understood. bacterial taxonomic analyses (assays described below). We hypothesized that specific Fusobacterium subspecies prevalent in colorectal cancer patients play critical roles in DNA isolation and bacterial 16S rDNA sequencing promoting proinflammatory response and neoplastic Total DNA was extracted from the frozen tissue speci- development. To test this hypothesis, we profiled the mens using a QIAamp kit (QIAGEN) according to the differential microbiomes in human colorectal cancers and manufacturer's instructions and stored at À20C prior to adjacent mucosal tissue by high-throughput bacterial 16S sequencing analysis. Microbial 16S ribosomal RNA (rRNA) rDNA sequencing. Then, using the Fusobacterium 16S gene- gene sequencing was performed as described previously targeted sequencing, we identified the specific Fusobacter- (30) with minor modifications. Briefly, the V4 region of ium spp. and subspecies associated with colorectal