Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma

Abstract

An estimated 15% or more of the cancer burden worldwide is attributable to known infectious agents. We screened colorectal carcinoma and matched normal tissue specimens using RNA-seq followed by host sequence subtraction and found marked over-representation of Fusobacterium nucleatum sequences in tumors relative to control specimens. F. nucleatum is an invasive anaerobe that has been linked previously to periodontitis and appendicitis, but not to cancer. Fusobacteria are rare constituents of the fecal microbiota, but have been cultured previously from biopsies of inflamed gut mucosa. We obtained a Fusobacterium isolate from a frozen tumor specimen; this showed highest sequence similarity to a known gut mucosa isolate and was confirmed to be invasive. We verified overabundance of Fusobacterium sequences in tumor versus matched normal control tissue by quantitative PCR analysis from a total of 99 subjects ( p = 2.5 3 10 -6), and we observed a positive association with lymph node metastasis.[Supplemental material is available for this article.] There are variations on the method, but the basic approach involves shotgun sequencing bulk DNA or RNA isolated from disease tissue, computational subtraction of all sequence reads recognized as human, and comparison of the residual reads to databases of known microbial sequences in order to identify microbial species present in the initial specimen. The method is complementary to traditional culture and histolology-based protocols, and new massively parallel sequencing technologies impart high sensitivity. At present the power of the method remains restricted by the content of microbial sequence databases, but with our increasing reach into microbial sequence space, the comprehensiveness of these data resources continues to improve. In oncology, the identification of a novel polyomavirus in Merkel Cell carcinoma (Feng et al. 2008) is a recent demonstration of the utility of a metagenomics approach.Colorectal carcinoma (CRC) is the fourth leading cause of cancer deaths, responsible for approximately 610,000 deaths per year worldwide (World Health Organization 2011). It is also one of the first and best genetically characterized cancers, and specific somatic mutations in oncogenes and tumor suppressor genes have been found that are associated with progression from adenomatous lesions (polyps) to invasive carcinoma (Vogelstein et al. 1988). The root cause of CRC is unclear, but inflammation is a well-recognized risk factor (Wu et al. 2009;McLean et al. 2011). Given the link between H. pylori-mediated inflammation and gastric cancer (Marshall and Warren 1984), we asked if inflammatory microorganisms are associated with other gastrointestinal (GI) cancers. We began to address this question by undertaking a metagenomic survey of colorectal carcinoma. ResultsTotal RNA was isolated from frozen sections of 11 matched pairs of colorectal carcinoma and adjacent normal tissue specimens. RNA was purified by host ribosomal sequence depletion, rather than poly(A) selection, in order to re...

Fusobacterium nucleatum associates with stages of colorectal neoplasia development, colorectal cancer and disease outcome

“…The results of this study confirm for the first time in European cohorts previous reports from North America [20,21] that Fn is over-represented in tumour tissue compared to normal tissue in colorectal cancers. More strikingly this work indicates that Fn load increases with disease progression from adenoma to cancer and may also be related to survival from cancer.…”

“…Previously published primer and probes were used [20,32] were assessed by binary logistic regression to assess influence of Fn levels on disease group. Further, associations between Fn levels and disease progression were analysed by Spearman correlation analysis.…”

“…showed over-representation of Fusobacterium nucleatum (Fn) in CRC tumours versus surrounding normal tissue [20,21]. Fn is a highly invasive, gram-negative anaerobic bacterium and part of the oral and gut commensal flora [22] that has been linked to several diseases, such as periodontitis [23], appendicitis [24], Lemierre's disease [25], and inflammatory bowel disease [26].…”

“…Fn is a highly invasive, gram-negative anaerobic bacterium and part of the oral and gut commensal flora [22] that has been linked to several diseases, such as periodontitis [23], appendicitis [24], Lemierre's disease [25], and inflammatory bowel disease [26]. Fn may contribute to CRC development by invading colonic mucosa and inducing local inflammation and increased expression of cytokines, leading to colorectal disease [20,[26][27][28][29]. More convincing evidence that Fn infection directly contributes to colorectal carcinogenesis rather than being a consequence of disease progression derives from two recent reports showing that Fn invasion, through its unique FadA adhesion, recruits tumour-infiltrating immune cells and generates an oncogenic/pro-inflammatory microenvironment conducive for colorectal neoplasia [30,31].…”

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

“…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…”