Fusobacterium nucleatum infection modulates the transcriptome and epigenome of HCT116 colorectal cancer cells in an oxygen-dependent manner.

Fusobacterium nucleatum, a gram-negative oral bacterium, has been consistently validated as a strong contributor to the progression of several types of cancer, including colorectal (CRC) and pancreatic cancer. While previous in vitro studies have shown that intracellular F. nucleatum enhances malignant phenotypes such as cell migration, the dependence of this regulation on features of the tumor microenvironment (TME) such as oxygen levels are wholly uncharacterized. Here we examine the influence of hypoxia in facilitating F. nucleatum invasion and its effects on host responses focusing on changes in the global epigenome and transcriptome. Using a multiomic approach, we analyze epigenomic alterations of H3K27ac and global transcriptomic alterations sustained within a hypoxia and normoxia conditioned CRC cell line HCT116 at 24 h following initial infection with F. nucleatum. Our findings reveal that intracellular F. nucleatum activates signaling pathways and biological processes in host cells similar to those induced upon hypoxia conditioning in the absence of infection. Furthermore, we show that a hypoxic TME favors F. nucleatum invasion and persistence and therefore infection under hypoxia may amplify malignant transformation by exacerbating the effects induced by hypoxia alone. These results motivate future studies to investigate host-microbe interactions in tumor tissue relevant conditions that more accurately define parameters for targeted cancer therapies.

Fusobacterium nucleatum induces oxaliplatin resistance by inhibiting ferroptosis through E-cadherin/ß-catenin/GPX4 axis in colorectal cancer.

Fusobacterium (F.) nucleatum is a carcinogenesis microbiota in colorectal cancer (CRC). Growing evidence shows that F. nucleatum contributes to chemoresistance. Ferroptosis is reported to restore the susceptibility of resistant cells to chemotherapy. However, the role of gut microbiota affecting ferroptosis in chemoresistance remains unclear. Here, we examined the CRC tissues of patients using 16S rRNA sequencing to investigate the possible connection between gut microbiota dysbiosis and the relapse of CRC. We found that a high abundance of F. nucleatum in CRC tissue is associated with relapse. We further demonstrated that F. nucleatum induced oxaliplatin resistance in vitro and in vivo. The transcriptome of an F. nucleatum-infected cell revealed ferroptosis was associated with F. nucleatum infection. We perform malondialdehyde, ferrous iron, and glutathione assays to verify the effect of F. nucleatum on ferroptosis under oxaliplatin treatment in vivo and in vitro. Mechanistically, F. nucleatum promoted oxaliplatin resistance by overexpressing GPX4 and then inhibiting ferroptosis. E-cadherin/ß-catenin/TCF4 pathway conducted the GPX4 overexpression effect of F. nucleatum. The chromatin immuno-precipitation quantitative PCR (CHIP-qPCR) and dual-luciferase reporter assay showed that F. nucleatum promoted TCF4 binding with GPX4. We also determined the E-cadherin/ß-catenin/TCF4/GPX4 axis related to tumor tissue F. nucleatum status and CRC relapse clinically. Here, we revealed the contribution of F. nucleatum to oxaliplatin resistance by inhibiting ferroptosis in CRC. Targeting F. nucleatum and ferroptosis will provide valuable insight into chemoresistance management and may improve outcomes for patients with CRC.

Effects of metronidazole on colorectal cancer occurrence and colorectal cancer liver metastases by regulating Fusobacterium nucleatum in mice.

OBJECTIVE: Colorectal cancer (CRC) represents a leading cause of cancer-related deaths. Metronidazole (MNZ) is exceedingly implicated in CRC. This study explored the roles of MNZ in mouse CRC occurrence and liver metastasis (CRLM). METHODS: Male BALB/c nude mice were subjected to CRC and CRLM modeling, orally administration with MNZ (1 g/L) 1 week before modeling, and disease activity index (DAI) evaluation. Fresh stool and anal swab samples were collected on the morning of the 28th day after modeling. The relative expression of Fusobacterium nucleatum (F. nucleatum) DNA was assessed by quantitative polymerase chain reaction. After euthanasia, tumor tissues and liver tissues were separated and the tumor volume and weight change were measured. The liver tissues were stained with hematoxylin-eosin to quantitatively analyze the metastatic liver nodules. Malignant tumor biomarker Ki67 protein levels in liver tissues/DNA from stool samples were detected by immunohistochemistry/high-throughput 16S rRNA gene sequencing. Bioinformatics analysis was performed on the raw sequence data to analyze microbial community richness (Chao1 index, ACE index) and microbial community diversity (Shannon index). RESULTS: The DAI and F. nucleatum DNA relative expression in feces and anal swabs of the CRC and CRLM groups were raised and repressed after MNZ intervention. MNZ repressed tumor occurrence and growth in mice to a certain extent, alleviated CRLM malignant degree (reduced liver metastases and Ki67-positive cell density/number), and suppressed CRC liver metastasis by regulating intestinal flora structure, which affected the intestinal characteristic flora of CRC and CRLM mice. CONCLUSION: MNZ suppressed CRC occurrence and CRLM in mice by regulating intestinal F. nucleatum.

Fusobacterium Nucleatum Is a Risk Factor for Metastatic Colorectal Cancer.

OBJECTIVE: Increasing evidence has indicated that there is a correlation between Fusobacterium nucleatum (F. nucleatum) abundance and poor prognosis of colorectal cancer (CRC). Furthermore, tumor metastasis plays a decisive role in the prognosis of CRC patients. Therefore, it was hypothesized that the abundance of F. nucleatum in CRC tissues affects the tumor metastasis. METHODS: In the present study, F. nucleatum DNA obtained from 141 resected CRC samples was quantified by qPCR to determine whether there were differences in F. nucleatum abundance between groups with and without CRC metastasis. RESULTS: The results revealed that F. nucleatum was more abundant in CRC patients with metastasis, and CRC tissues enriched with F. nucleatum had a higher risk of lymph node metastasis and distant metastasis. The receiver operating characteristic curve indicated that F. nucleatum in CRC tissues could be used as an indicator for CRC metastasis, to some extent. Furthermore, the in vitro experiments (electron microscopy, and migration and invasion trials) revealed that F. nucleatum was a highly invasive bacterial strain, and could significantly enhance the invasion and migration capacity of SW480 and SW620 cells. In addition, a meta-analysis comprehensively indicated a slight correlation between F. nucleatum abundance and advanced CRC stage (RR=1.17, 95% CI: 1.00-1.37, P=0.04, random effect). CONCLUSION: There is a correlation between F. nucleatum abundance and CRC metastasis, and F. nucleatum may serve as a metastasis biomarker for CRC patients.

Detection of Fusobacterium nucleatum DNA in primary care patient stool samples does not predict progression of colorectal neoplasia.

BACKGROUND: Carriage of certain bacterial species may represent potential biomarkers of colorectal cancer (CRC). Prominent among these is Fusobacterium nucleatum. We explored the association of F. nucleatum DNA in stool samples with the presence of colonic neoplastic lesions in a cohort of primary care patients, and compared our findings with those from an unrelated cohort of colonoscopy patients followed clinically over time. METHODS: Carriage rates of F. nucleatum in stool samples were assessed in 185 patients referred for a faecal immunochemical test (FIT) by their general practitioners (GPs). Comparisons were made with stool samples from 57 patients diagnosed with CRC and 57 age-matched healthy controls, and with tissue samples taken at colonoscopy from 150 patients with a decade of subsequent clinical follow-up. FINDINGS: F. nucleatum DNA was found at a high rate (47.0%) in stool samples from primary care patients, and more often in stool samples from CRC patients (47.4%) than in healthy controls (7.0%), (P = 7.66E-7). No association was found between carriage of F. nucleatum and FIT positivity (P = 0.588). While evidence of stool-associated F. nucleatum DNA was significantly more likely to indicate a lesion in those primary care patients progressed to colonoscopy (P = 0.023), this finding did not extend to the progression of neoplastic lesions in the 150 patients with a decade of follow up. CONCLUSION: The finding of F. nucleatum DNA at similar rates in stool samples from patients diagnosed with CRC and in primary care patients with pre-cancerous lesions supports growing awareness that the presence of these bacteria may be a biomarker for increased risk of disease. However, molecular evidence of F. nucleatum did not predict progression of colonic lesions, which may lessen the utility of this bacterium as a biomarker for increased risk of disease.

Effect of the Progression of Fusobacterium nucleatum-induced Apical Periodontitis on the Gut Microbiota.

INTRODUCTION: Fusobacterium nucleatum, which is involved in the development of periodontal disease and apical lesions, can be transmitted to the colon and metastasize to colorectal cancer, suggesting a link between oral and systemic diseases. We analyzed the effects of F. nucleatum on bacterial flora in the gut and surrounding organs in a rat model of apical periodontitis and analyzed the infection route to the gut and distant organs. METHODS: We induced apical periodontitis in rat molars by infecting the dental pulp with F. nucleatum and then took X-ray images and performed histopathologic analyses. Next, we removed the maxilla, gut, heart, liver, and kidney from the rats at 0, 2, 4, and 8 weeks postsurgery and then extracted DNA samples and performed polymerase chain reaction and microbiome analyses using the Illumina MiSeq (Illumina Co, Tokyo, Japan). RESULTS: The presence of inflammatory cell infiltration confirmed apical periodontitis from 2-8 weeks. Polymerase chain reaction and microbiome analyses revealed F. nucleatum in the rat gut from 2 weeks and in the kidney from 8 weeks. The rat gut, heart, liver, and kidney exhibited altered bacterial flora, including a marked decrease in Verrucomicrobia and an increase in Proteobacteria after 2 weeks and increases in Bacteroidetes and Firmicutes after 4 weeks. CONCLUSIONS: The onset of F. nucleatum-induced apical periodontitis changed the bacterial flora in the rat gut, heart, liver, and kidney, with a confirmed progressing infection in the large intestines.

Modulation of the Host Cell Transcriptome and Epigenome by Fusobacterium nucleatum.

Fusobacterium nucleatum is a ubiquitous opportunistic pathogen with an emerging role as an oncomicrobe in colorectal cancer and other cancer settings. F. nucleatum can adhere to and invade host cells in a manner that varies across F. nucleatum strains and host cell phenotypes. Here, we performed pairwise cocultures between three F. nucleatum strains and two immortalized primary host cell types (human colonic epithelial [HCE] cells and human carotid artery endothelial [HCAE] cells) followed by transcriptome sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) to investigate transcriptional and epigenetic host cell responses. We observed that F. nucleatum-induced host cell transcriptional modulation involves strong upregulation of genes related to immune migration and inflammatory processes, such as TNF, CXCL8, CXCL1, and CCL20. Furthermore, we identified genes strongly upregulated in a cell line-specific manner. In HCE cells, overexpressed genes included UBD and DUOX2/DUOXA2, associated with p53 degradation-mediated proliferation and intestinal reactive oxygen species (ROS) production, respectively. In HCAE cells, overexpressed genes included EFNA1 and LIF, two genes commonly upregulated in colorectal cancer and associated with poor patient outcomes, and PTGS2 (COX2), a gene associated with the protective effect of aspirin in the colorectal cancer setting. Interestingly, we also observed downregulation of numerous histone modification genes upon F. nucleatum exposure. We used the ChIP-seq data to annotate chromatin states genome wide and found significant chromatin remodeling following F. nucleatum exposure in HCAE cells, with increased frequencies of active enhancer and low-signal/quiescent states. Thus, our results highlight increased inflammation and chemokine gene expression as conserved host cell responses to F. nucleatum exposure and extensive host cell epigenomic changes specific to host cell type. IMPORTANCE Fusobacterium nucleatum is a bacterium normally found in the healthy oral cavity but also has an emerging role in colorectal cancer and other cancer settings. The host-microbe interactions of F. nucleatum and its involvement in tumor initiation, progression, and treatment resistance are not fully understood. We explored host cell changes that occur in response to F. nucleatum. We identified key genes differentially expressed in response to various conditions of F. nucleatum exposure and determined that the conserved host cell response to F. nucleatum was dominated by increased inflammation and chemokine gene expression. Additionally, we found extensive host cell epigenomic changes as a novel aspect of host modulation associated with F. nucleatum exposure. These results extend our understanding of F. nucleatum as an emerging pathogen and highlight the importance of considering strain heterogeneity and host cell phenotypic variation when exploring pathogenic mechanisms of F. nucleatum.

Conversion from epithelial to partial-EMT phenotype by Fusobacterium nucleatum infection promotes invasion of oral cancer cells.

The ability of cancer cells to undergo partial-epithelial mesenchymal transition (p-EMT), rather than complete EMT, poses a higher metastatic risk. Although Fusobacterium nucleatum mainly inhabits in oral cavity, attention has been focused on the F. nucleatum involvement in colorectal cancer development. Here we examined the p-EMT regulation by F. nucleatum in oral squamous cell carcinoma (OSCC) cells. We cultured OSCC cells with epithelial, p-EMT or EMT phenotype with live or heat-inactivated F. nucleatum. Expression of the genes involved in epithelial differentiation, p-EMT and EMT were examined in OSCC cells after co-culture with F. nucleatum by qPCR. Cell growth and invasion of OSCC cells were also examined. Both live and heat-inactivated F. nucleatum upregulated the expression of p-EMT-related genes in OSCC cells with epithelial phenotype, but not with p-EMT or EMT phenotype. Moreover, F. nucleatum promoted invasion of OSCC cells with epithelial phenotype. Co-culture with other strains of bacteria other than Porphyromonas gingivalis did not alter p-EMT-related genes in OSCC cells with epithelial phenotype. F. nucleatum infection may convert epithelial to p-EMT phenotype via altering gene expression in OSCC. Oral hygiene managements against F. nucleatum infection may contribute to reduce the risk for an increase in metastatic ability of OSCC.

Genetic and molecular determinants of polymicrobial interactions in Fusobacterium nucleatum.

A gram-negative colonizer of the oral cavity, Fusobacterium nucleatum not only interacts with many pathogens in the oral microbiome but also has the ability to spread to extraoral sites including placenta and amniotic fluid, promoting preterm birth. To date, however, the molecular mechanism of interspecies interactions-termed coaggregation-by F. nucleatum and how coaggregation affects bacterial virulence remain poorly defined. Here, we employed genome-wide transposon mutagenesis to uncover fusobacterial coaggregation factors, revealing the intertwined function of a two-component signal transduction system (TCS), named CarRS, and a lysine metabolic pathway in regulating the critical coaggregation factor RadD. Transcriptome analysis shows that CarR modulates a large regulon including radD and lysine metabolic genes, such as kamA and kamD, the expression of which are highly up-regulated in the ΔcarR mutant. Significantly, the native culture medium of ΔkamA or ΔkamD mutants builds up abundant amounts of free lysine, which blocks fusobacterial coaggregation with streptococci. Our demonstration that lysine-conjugated beads trap RadD from the membrane lysates suggests that lysine utilizes RadD as its receptor to act as a metabolic inhibitor of coaggregation. Lastly, using a mouse model of preterm birth, we show that fusobacterial virulence is significantly attenuated with the ΔkamA and ΔcarR mutants, in contrast to the enhanced virulence phenotype observed upon diminishing RadD (ΔradD or ΔcarS mutant). Evidently, F. nucleatum employs the TCS CarRS and environmental lysine to modulate RadD-mediated interspecies interaction, virulence, and nutrient acquisition to thrive in the adverse environment of oral biofilms and extraoral sites.

Fusobacterium watanabei sp. nov. As additional species within the genus Fusobacerium, isolated from human clinical specimens.

Eight spindle-shaped bacteria were isolated from clinical samples in Japan and investigated for their taxonomic position. Phylogenetic trees (based on 16S rRNA, rpoB, zinc protease, and gyrB gene sequence comparisons) showed distinct clustering of eight strains with the type strain of Fusobacterium nucleatum and its closely related species. In silico whole genome comparison analysis based on average nucleotide index based on BLAST (ANIb) and digital DNA-DNA hybridization (dDDH) data between our clinical isolates (PAGU 1795, PAGU 1796T, and PAGU 1797) and the type strain of the closely related species showed values of less than 92.4% and 49.5%, respectively. On the basis of its phylogenetic and genomic distinctiveness together with differential phenotypic properties and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) characteristic signal patterns, we propose Fusobacterium watanabei sp. nov., with the type strain PAGU 1796T (= GTC 21791T = CCUG 74246T).

F. nucleatum targets lncRNA ENO1-IT1 to promote glycolysis and oncogenesis in colorectal cancer.

OBJECTIVE: Microbiota disorder promotes chronic inflammation and carcinogenesis. High glycolysis is associated with poor prognosis in patients with colorectal cancer (CRC). However, the potential correlation between the gut microbiota and glucose metabolism is unknown in CRC. DESIGN: 18F-FDG (18F-fluorodeoxyglucose) PET (positron emission tomography)/CT image scanning data and microbiota PCR analysis were performed to measure the correlation between metabolic alterations and microbiota disorder in 33 patients with CRC. Multiple colorectal cancer models, metabolic analysis and Seahorse assay were established to assess the role of long non-coding RNA (lncRNA) enolase1-intronic transcript 1 (ENO1-IT1) in Fusobacterium (F.) nucleatum-induced glucose metabolism and colorectal carcinogenesis. RNA immunoprecipitation and chromatin immunoprecipitation sequencing were conducted to identify potential targets of lncRNA ENO1-IT1. RESULTS: We have found F. nucleatum abundance correlated with high glucose metabolism in patients with CRC. Furthermore, F. nucleatum supported carcinogenesis via increasing CRC cell glucose metabolism. Mechanistically, F. nucleatum activated lncRNA ENO1-IT1 transcription via upregulating the binding efficiency of transcription factor SP1 to the promoter region of lncRNA ENO1-IT1. Elevated ENO1-IT behaved as a guider modular for KAT7 histone acetyltransferase, specifying the histone modification pattern on its target genes, including ENO1, and consequently altering CRC biological function. CONCLUSION: F. nucleatum and glucose metabolism are mechanistically, biologically and clinically connected to CRC. Targeting ENO1 pathway may be meaningful in treating patients with CRC with elevated F. nucleatum.

New Roles for Fusobacterium nucleatum in Cancer: Target the Bacteria, Host, or Both?

Fusobacterium nucleatum is an oral bacterium associated with colorectal cancer (CRC) proliferation, chemoresistance, inflammation, metastasis, and now DNA damage. While controlling F. nucleatum through antibiotics could reduce cancer severity, this article proposes additional strategies to block Fusobacterium-host interactions, as well as treatment of activated host immune and oncogenic signaling pathways in CRC.

The DNA Glycosylase NEIL2 Suppresses Fusobacterium-Infection-Induced Inflammation and DNA Damage in Colonic Epithelial Cells.

Colorectal cancer (CRC) is the third most prevalent cancer, while the majority (80-85%) of CRCs are sporadic and are microsatellite stable (MSS), and approximately 15-20% of them display microsatellite instability (MSI). Infection and chronic inflammation are known to induce DNA damage in host tissues and can lead to oncogenic transformation of cells, but the role of DNA repair proteins in microbe-associated CRCs remains unknown. Using CRC-associated microbes such as Fusobacterium nucleatum (Fn) in a coculture with murine and human enteroid-derived monolayers (EDMs), here, we show that, among all the key DNA repair proteins, NEIL2, an oxidized base-specific DNA glycosylase, is significantly downregulated after Fn infection. Fn infection of NEIL2-null mouse-derived EDMs showed a significantly higher level of DNA damage, including double-strand breaks and inflammatory cytokines. Several CRC-associated microbes, but not the commensal bacteria, induced the accumulation of DNA damage in EDMs derived from a murine CRC model, and Fn had the most pronounced effect. An analysis of publicly available transcriptomic datasets showed that the downregulation of NEIL2 is often encountered in MSS compared to MSI CRCs. We conclude that the CRC-associated microbe Fn induced the downregulation of NEIL2 and consequent accumulation of DNA damage and played critical roles in the progression of CRCs.

Knock down of BMSC-derived Wnt3a or its antagonist analogs attenuate colorectal carcinogenesis induced by chronic Fusobacterium nucleatum infection.

By establishing the Fusobacterium nucleatum (F. nucleatum) infected-bone mesenchymal stem cells (BMSCs) transplantation model in APCMin/+ mice, we investigated the role of BMSCs in the development of intestinal tumors induced by F. nucleatum. ApcMin/++F. nucleatum + BMSCs mice showed increased susceptibility to intestinal tumors and accelerated tumor growth. BMSCs could also enhance tumor-initiating capability, invasive traits after F. nucleatum infection in vitro, and tumorigenicity in a nude murine model. Mechanistically, BMSCs were recruited to the submucosa, migrated to the mucosal layer, and might activate the canonical Wnt/ß-catenin/TGIF axis signaling. Further mechanistic results illustrated increased production of the Wnt3a protein was found in ApcMin/++F. nucleatum + BMSCs mice, and BMSCs were likely the major source of Wnt3a. Intriguingly, a deletion of Wnt3a via BMSC interference or antagonist analogs led to a significantly attenuated capacity of ApcMin/++F. nucleatum mice to generate intestinal tumors. The findings suggest that BMSCs have the potential to migrate and accelerate F. nucleatum-induced colorectal tumorigenesis by modulating Wnt3a secretion; knockdown of BMSC-derived Wnt3a or antagonist analogs could attenuate carcinogenesis. Thus, Wnt3a might be a potential pharmaceutical target for the prevention and treatment of F. nucleatum-related colorectal cancer.

Fusobacterium nucleatum promotes epithelial-mesenchymal transiton through regulation of the lncRNA MIR4435-2HG/miR-296-5p/Akt2/SNAI1 signaling pathway.

Fusobacterium nucleatum, an anaerobic oral opportunistic pathogen associated with periodontitis, has been considered to be associated with the development of oral squamous cell carcinoma (OSCC). However, the initial host molecular alterations induced by F. nucleatum infection which may promote predisposition to malignant transformation through epithelial-mesenchymal transition (EMT) have not yet been clarified. In the present study, we monitored the ability of F. nucleatum to induce EMT-associated features, and our results showed that F. nucleatum infection promoted cell migration in either noncancerous human immortalized oral epithelial cells (HIOECs) or the two OSCC cell lines SCC-9 and HSC-4, but did not accelerate cell proliferation or cell cycle progression. Mesenchymal markers, including N-cadherin, Vimentin, and SNAI1, were upregulated, while E-cadherin was decreased and was observed to translocate to the cytoplasm. Furthermore, FadA adhesin and heat-inactivated F. nucleatum were found to cause a similar effect as the viable bacterial cells. The upregulated lncRNA MIR4435-2HG identified by the high-throughput sequencing was demonstrated to negatively regulate the expression of miR-296-5p, which was downregulated in F. nucleatum-infected HIOECs and SCC-9 cells. The binding of MIR4435-2HG and miR-296-5p was validated via a dual-luciferase reporter assay. Additionally, knockdown of MIR4435-2HG with siRNA leads to a decrease in SNAI1 expression, while miR-296-5p could further negatively and indirectly regulate SNAI1 expression via Akt2. Therefore, our study demonstrated that F. nucleatum infection could trigger EMT via lncRNA MIR4435-2HG/miR-296-5p/Akt2/SNAI1 signaling pathway, and EMT process may be a probable link between F. nucleatum infection and initiation of oral epithelial carcinomas.

Association Between Fusobacterium nucleatum, Pathway Mutation, and Patient Prognosis in Colorectal Cancer.

BACKGROUND: There is a close link between Fusobacterium nucleatum and colorectal cancer (CRC) tumorigenesis and chemoresistance. However, the genetic characteristics and clinical significance of CRC related with F. nucleatum remains unclear. This study evaluated the relationship between F. nucleatum, pathway mutation, and patient prognosis. METHODS: Fusobacterium nucleatum amount in the tumor tissue and adjacent normal tissue were measured by quantitative polymerase chain reaction in adjuvant (N = 128) and metastatic (N = 118) cohorts. Patients were divided into binary (F. nucleatum-high and F. nucleatum-low) according to F. nucleatum amount. Targeted next-generation sequencing of 40 genes included in the 5 critical pathways (WNT, P53, RTK-RAS, PI3 K, and TGF-ß) was performed in the adjuvant cohort. RESULTS: Patients with MSI-H and CIMP-H had higher amount of F. nucleatum in tumor tissue. Fusobacterium nucleatum-high patients had higher rates of transition mutation and C to T (G to A) nucleotide change regardless of MSI status. In addition, mutation rate of AMER1 and ATM genes, and TGF-ß pathway was higher in F. nucleatum-high patients. Fusobacterium nucleatum-high was associated with poor overall survival (OS) in the palliative cohort (26.4 vs. 30.7 months, p = 0.042). Multivariate analysis revealed F. nucleatum-high as an independent negative prognostic factor for OS [adjusted hazard ratio of 1.69 (95% confidence interval 1.04-2.75), p = 0.034]. However, F. nucleatum amount was not associated with recurrence in the adjuvant cohort. CONCLUSIONS: F. nucleatum-high was associated with poor survival in metastatic CRC. In addition, we identified mutational characteristics of colorectal cancer according to F. nucleatum amount.

Replication Study: Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma.

As part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Repass et al., 2016), that described how we intended to replicate an experiment from the paper 'Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma' (Castellarin et al., 2012). Here we report the results. When measuring Fusobacterium nucleatum DNA by qPCR in colorectal carcinoma (CRC), adjacent normal tissue, and separate matched control tissue, we did not detect a signal for F. nucleatum in most samples: 25% of CRCs, 15% of adjacent normal, and 0% of matched control tissue were positive based on quantitative PCR (qPCR) and confirmed by sequencing of the qPCR products. When only samples with detectable F. nucleatum in CRC and adjacent normal tissue were compared, the difference was not statistically significant, while the original study reported a statistically significant increase in F. nucleatum expression in CRC compared to adjacent normal tissue (Figure 2; Castellarin et al., 2012). Finally, we report a meta-analysis of the result, which suggests F. nucleatum expression is increased in CRC, but is confounded by the inability to detect F. nucleatum in most samples. The difference in F. nucleatum expression between CRC and adjacent normal tissues was thus smaller than the original study, and not detected in most samples.

Fusobacterium nucleatum as a prognostic marker of colorectal cancer in a Japanese population.

BACKGROUND: Accumulating evidence shows an overabundance of Fusobacterium nucleatum in colorectal tumor tissues. However, the correlation between the absolute copy number of F. nucleatum in colorectal cancer tissues and colorectal cancer progression is unclear from previous reports. Therefore, we performed a study to compare the abundance of F. nucleatum in colorectal tissues with clinicopathologic and molecular features of colorectal cancer. METHODS: We collected 100 colorectal cancer tissues and 72 matched normal-appearing mucosal tissues. Absolute copy numbers of F. nucleatum were measured by droplet digital PCR. RESULTS: The detection rates of F. nucleatum were 63.9% (46/72) in normal-appearing mucosal tissues and 75.0% (75/100) in CRC tissue samples. The median copy number of F. nucleatum was 0.4/ng DNA in the normal-appearing colorectal mucosa in patients with colorectal cancer and 1.9/ng DNA in the colorectal cancer tissues (P = 0.0031). F. nucleatum copy numbers in stage IV colorectal cancer tissues were significantly higher than those in the normal-appearing mucosa in patients with colorectal cancer (P = 0.0016). The abundance of F. nucleatum in colorectal cancer tissues correlated with tumor size and KRAS mutation and was significantly associated with shorter overall survival times; this trend was notable in the patients with stage IV colorectal cancer. Focusing on normal-appearing mucosa in the patients with colorectal cancer, the F. nucleatum copy number was significantly higher in the patients with stage IV rather than stages I-III. CONCLUSION: These results suggest that determining F. nucleatum levels may help predict clinical outcomes in colorectal cancer patients. Further confirmatory studies using independent datasets are required to confirm our findings.