Purpose:Fusobacterium nucleatum (F. nucleatum) is a component of the human microbiome that primarily inhabits the oral cavity. It causes periodontal disease and has also been implicated in the development of human cancers. Although there are several reports of the relationship between F. nucleatum and the clinical outcome in human cancers, its prognostic significance in esophageal cancer remains unclear.

Experimental Design: We quantified F. nucleatum DNA in 325 resected esophageal cancer specimens by qPCR. Significant pathways in F. nucleatum–positive esophageal cancer tissues were identified by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis using microarray data.

Results: Esophageal cancer tissues contained significantly more F. nucleatum DNA than matched normal esophageal mucosa (P = 0.021; n = 60). F. nucleatum DNA was detected in 74 of 325 cases (23%). F. nucleatum DNA positivity was significantly associated with tumor stage, but not with sex, age, performance status, tobacco use, alcohol use, histology, tumor location, or preoperative treatment. F. nucleatum DNA positivity was also significantly associated with cancer-specific survival [log-rank P = 0.0039; univariate HR = 2.01; 95% confidence interval (CI), 1.22–3.23; P = 0.0068; multivariate HR = 1.78; 95% CI, 1.06–2.94; P = 0.031]. The top-ranked KEGG pathway in F. nucleatum–positive tissues was “cytokine–cytokine receptor interaction.” A significant relationship between F. nucleatum and the chemokine CCL20 was validated by IHC.

Conclusions:F. nucleatum in esophageal cancer tissues was associated with shorter survival, suggesting a potential role as a prognostic biomarker. F. nucleatum might also contribute to aggressive tumor behavior through activation of chemokines, such as CCL20. Clin Cancer Res; 22(22); 5574–81. ©2016 AACR.

This article is featured in Highlights of This Issue, p. 5395

Translational Relevance

Fusobacterium nucleatum (F. nucleatum), which primarily inhabits the oral cavity, causes periodontal disease. F. nucleatum influences the development and progression of colorectal cancer. Furthermore, the presence of F. nucleatum is associated with a poor prognosis in patients with colorectal cancer. However, no studies to date have examined the prognostic impact of F. nucleatum in esophageal cancer. In this study, we quantified F. nucleatum DNA in 325 resected esophageal cancer specimens by qPCR. This is the first study to provide the evidence for the relationship between F. nucleatum and poor prognosis in esophageal cancer. In addition, using KEGG enrichment analysis, we demonstrated that F. nucleatum might contribute to the acquisition of aggressive tumor behavior through the activation of chemokines, such as CCL20. Our data suggest that F. nucleatum DNA status can have a potential role as a prognostic biomarker.

Esophageal cancer is the fifth most common cause of cancer-related death in men and the eighth most common in women worldwide (1). Despite the development of multimodal therapies, including surgery, chemotherapy, radiotherapy, and chemoradiotherapy, the prognosis of patients, including those who undergo complete resection, remains poor (2–4). Further studies are therefore needed to clarify the pathogenesis of esophageal cancer and to explore new diagnostic and therapeutic possibilities. In addition, the identification of new prognostic or predictive markers for esophageal cancer could improve the use of risk-adapted treatment strategies and help to stratify patients for drugs targeting these tumor characteristics in future clinical trials.

Research into the microbiome is a rapidly advancing field in human cancers (5–7). More than 100 trillion bacteria inhabit the human body and form their own flora (microbiome) in individual organs. The gut microbiome has recently been shown to play an important role in health, as well as in diseases, such as obesity (8, 9), inflammatory bowel disease (10, 11), diabetes (12, 13), nonalcoholic fatty liver disease (14, 15), and several types of cancers. Fusobacterium nucleatum (F. nucleatum; a non–spore-forming, anaerobic gram-negative bacterium) is part of the normal flora in the human oral cavity, vagina, and gastrointestinal mucosa (16). It is recognized as a pathogen in periodontal diseases (17), chorioamnionitis (18), and inflammatory bowel disease (10, 19). Regarding the association between F. nucleatum and gastroenterological cancers, metagenomic analysis has shown an overabundance of F. nucleatum in colorectal cancer tissues (20, 21). F. nucleatum has also been shown to activate the WNT/β-catenin signaling pathway in colorectal cancer cells, and to potentially promote tumor growth (22). Recent studies reported that high levels of F. nucleatum DNA were linked to a poor prognosis in human cancers (23, 24), whereas others reported no association between F. nucleatum DNA levels and patient survival (Supplementary Table S1; refs. 20, 25). However, no studies to date have examined the prognostic impact of F. nucleatum in esophageal cancer tissues.

In this study, we quantified F. nucleatum DNA in 325 samples of resected esophageal cancer by qPCR and examined its prognostic value. We also clarified the mechanism whereby F. nucleatum may confer a poor prognosis by enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The results of this study suggest that F. nucleatum may have a potential role as a prognostic biomarker in esophageal cancer.

Study group

We analyzed 325 formalin-fixed, paraffin-embedded (FFPE) esophageal cancer specimens from consecutive patients undergoing resection at Kumamoto University Hospital (Kumamoto, Japan) between April 2005 and June 2013. Tumor staging was carried out according to the American Joint Committee on Cancer Staging Manual (7th edition; ref. 26). The vast majority of cases were diagnosed as squamous cell carcinoma (SCC); there were 300 cases (92%) of SCC, 12 (3.7%) of adenocarcinoma, and 13 (4.0%) of others. A total of 117 received preoperative treatment [73 chemotherapy (cisplatin, 5-fluorouracil either with or without docetaxel) 44 chemoradiotherapy]. Patients were observed at 1- to 3-month intervals until death or January 31, 2016, whichever came first. Overall survival (OS) was defined as the time from the date of surgery to the date of death. Cancer-specific survival was defined as the time from the date of surgery and the date of death attributable to esophageal cancer. Written informed consent was obtained from each subject, and the study procedures were approved by the Institutional Review Board. The term “prognostic marker” is used throughout this article according to the REMARK guidelines (27).

DNA extraction and qPCR for F. nucleatum

Genomic DNA was extracted from FFPE esophageal cancer tissues using a QIAamp DNA FFPE Tissue Kit (Qiagen). We determined the amount of F. nucleatum DNA by qPCR assay. The nus G gene of F. nucleatum and the reference human gene SLCO2A1 were amplified using custom-made TaqMan primer/probe sets (Applied Biosystems). The primer and probe sequences for each Custom TaqMan Gene Expression Assay were as follows: F. nucleatum forward primer, 5′-TGGTGTCATTCTTCCAAAAATATCA-3′; F. nucleatum reverse primer, 5′-AGATCAAGAAGGACAAGTTGCTGAA-3′; F. nucleatum FAM probe, 5′-ACTTTAACTCTACCATGTTCA-3′; SLCO2A1 forward primer, 5′-ATCCCCAAAGCACCTGGTTT-3′; SLCO2A1 reverse primer, 5′-AGAGGCCAAGATAGTCCTGGTAA-3′; SLCO2A1 VIC probe, 5′-CCATCCATGTCCTCATCTC-3′. Assays were performed in a 384-well optical PCR plate. DNA was amplified and detected using a LightCycler 480 Instrument II (Roche) under the following reaction conditions: initial denaturation at 95°C for 10 minutes, 15 seconds at 95°C, and 60 seconds at 60°C. The amount of F. nucleatum DNA in each tissue was normalized relative to SLCO2A1 (28).

Immunohistochemical staining

FFPE tissue was serially sectioned at 3 to 5 μm, dewaxed, deparaffinized in xylene, and rehydrated through a series of graded alcohols. The samples were boiled for 15 minutes in a microwave oven in Histofine (pH = 9.0; Nichirei) to increase antigen retrieval. Endogenous peroxidases were blocked by 3% hydrogen peroxidase treatment for 30 minutes. The slides were incubated with primary antibody (1:50 dilution of rabbit mAb for macrophage inflammatory protein 3α (CC-chemokine cysteine motif chemokine ligand 20, CCL20; ab9829; Abcam) overnight at 4°C. Detection was performed with a biotin-free horseradish peroxidase–labeled polymer of the Envision Plus detection system (Dako). The sections were developed in 3,3-diaminobenzidine and counterstained with Mayer hematoxylin. The slides were then dehydrated through graded alcohols and covered with coverslips. Staining intensity and percentage of CCL20-positive tumor cells were assessed. The extent of staining was categorized semiquantitatively, based on the percentage of positive tumor cells: 0 (≤5% positive cells), 1 (6%–25% positive cells), 2 (26%–50% positive cells), 3 (51%–75% positive cells), and 4 (>75% positive cells). The intensities of cytoplasmic and membrane staining were also determined semiquantitatively as follows: 0 (negative), 1 (weakly positive), 2 (moderately positive), and 3 (strongly positive). The scores of sections were defined as “extent of staining × intensity.”

Microarray and KEGG pathway enrichment analysis

Total RNA was isolated from frozen sections of 10 esophageal cancer biopsy specimens using an RNeasy Mini Kit (Qiagen). Gene expression microarray analysis was carried out using SurePrint G3 Human GE Microarray 8 × 60K Ver. 2.0 (Agilent Technologies) according to the manufacturer's protocol. Differentially expressed genes (DEG) were screened by comparing the RNA expression levels of esophageal cancer specimens between F. nucleatum–positive and F. nucleatum–negative groups, using Subio Platform (Subio Inc.). KEGG pathway enrichment analyses were performed to identify the biological functions and pathways represented by the identified DEGs using the Database for Annotation, Visualization and Integrated Discovery 6.7 software (https://david.ncifcrf.gov/; ref. 29).

Statistical analysis

All statistical analyses were carried out using JMP, version 10 (SAS Institute). All P values were two-sided. We compared mean values using Student t tests for age and body mass index, and χ2 or Fisher exact tests for all other variables. Survival time distribution in the survival analysis was assessed by the Kaplan–Meier method using log-rank tests. We constructed a multivariate model to compute the HR based on the F. nucleatum DNA status, including sex (male vs. female), age at surgery (<65 vs. ≥65 years), year of surgery (2005–2009 vs. 2010–2013), tobacco use (yes vs. no), alcohol use (yes vs. no), performance status (0 vs. 1–), tumor location (upper vs. lower), tumor stage (I and II vs. III and IV), and preoperative treatment (absent vs. present). Interactions were assessed by including the cross-product of the F. nucleatum DNA status and another variable of interest in a Cox model.

F. nucleatum in esophageal cancer tissues

We assessed the relative amounts of F. nucleatum DNA in esophageal cancer tissues by qPCR assay. F. nucleatum DNA levels were higher in esophageal cancer tissues than in paired adjacent nontumor tissues (n = 60, P = 0.021; Fig. 1A). We also measured the relative F. nucleatum DNA levels in cancer tissues from the 325 esophageal cancer cases. F. nucleatum was detected in 74 (23%) of 325 cases (Fig. 1B). The relative F. nucleatum DNA content in esophageal cancer tissues ranged from 3.0 × 10−4−2.8 × 100 (median, 2.3 × 10−2).

Figure 1.

F. nucleatum expression in esophageal cancer. A,F. nucleatum expression in tumor and adjacent normal tissue samples in 60 patients with esophageal cancer. F. nucleatum expression was significantly higher in tumor than in adjacent normal tissue (P = 0.021). B,F. nucleatum expression status in 325 patients with esophageal cancer.

Figure 1.

F. nucleatum expression in esophageal cancer. A,F. nucleatum expression in tumor and adjacent normal tissue samples in 60 patients with esophageal cancer. F. nucleatum expression was significantly higher in tumor than in adjacent normal tissue (P = 0.021). B,F. nucleatum expression status in 325 patients with esophageal cancer.

Close modal

Relationship between tumor F. nucleatum DNA status and clinicopathologic features in esophageal cancer

The clinicopathologic features of the 325 cases according to F. nucleatum status are shown in Table 1. F. nucleatum positivity was not associated with age, sex, year of operation, preoperative performance status, smoking history, alcohol history, comorbidity, tumor location, histology, tumor size, or preoperative therapy (all P > 0.05), but was associated with tumor stage (P = 0.016), T stage (P < 0.01), and N stage (P = 0.039).

Table 1.

F. nucleatum DNA status in esophageal cancers and clinical and tumor features

Clinical or pathologic featureF. nucleatum DNA
NNegativePositiveP
All cases 325 251 74  
Mean age ± SD 65.9 ± 9.2 65.6 ± 9.5 66.7 ± 8.0 0.52 
Sex    1.0 
 Male 287 221 66  
 Female 38 30  
Year of operation    0.69 
 2005 to 2009 172 131 41  
 2010 - 153 120 33  
Performance status    1.0 
 0 252 194 58  
 1 - 73 57 16  
Tobacco use    0.86 
 Yes 271 210 61  
 No 54 41 13  
Alcohol use    0.72 
 Yes 274 210 64  
 No 51 41 10  
Comorbidity    0.48 
 Present 223 175 48  
 Absent 102 76 26  
Location    0.16 
 Upper 54 46  
 Lower 271 205 66  
Histology    0.64 
 SCC 300 230 70  
 Adeno 12 10  
 Others 13 11  
Tumor size ± SD 4.2 ± 3.5 3.9 ± 2.4 4.4 ± 1.8 0.12 
Stage    0.016 
 I 155 131 24  
 II 42 32 10  
 III 118 82 36  
 IV 10  
Preoperative treatment    0.053 
 Present 117 83 34  
 Absent 208 168 40  
Clinical or pathologic featureF. nucleatum DNA
NNegativePositiveP
All cases 325 251 74  
Mean age ± SD 65.9 ± 9.2 65.6 ± 9.5 66.7 ± 8.0 0.52 
Sex    1.0 
 Male 287 221 66  
 Female 38 30  
Year of operation    0.69 
 2005 to 2009 172 131 41  
 2010 - 153 120 33  
Performance status    1.0 
 0 252 194 58  
 1 - 73 57 16  
Tobacco use    0.86 
 Yes 271 210 61  
 No 54 41 13  
Alcohol use    0.72 
 Yes 274 210 64  
 No 51 41 10  
Comorbidity    0.48 
 Present 223 175 48  
 Absent 102 76 26  
Location    0.16 
 Upper 54 46  
 Lower 271 205 66  
Histology    0.64 
 SCC 300 230 70  
 Adeno 12 10  
 Others 13 11  
Tumor size ± SD 4.2 ± 3.5 3.9 ± 2.4 4.4 ± 1.8 0.12 
Stage    0.016 
 I 155 131 24  
 II 42 32 10  
 III 118 82 36  
 IV 10  
Preoperative treatment    0.053 
 Present 117 83 34  
 Absent 208 168 40  

Abbreviation: Adeno, adenocarcinoma.

Tumor F. nucleatum DNA status and patient survival

There were 112 deaths among the 325 esophageal cancer patients, including 75 esophageal cancer-specific deaths. The median follow-up time for censored patients was 2.6 years.

According to Kaplan–Meier analysis, F. nucleatum–positive patients had significantly shorter cancer-specific survival (log-rank P = 0.0039) and OS (log-rank P = 0.046) compared with F. nucleatum–negative cases (Fig. 2). We also analyzed F. nucleatum DNA status by Cox regression analysis. F. nucleatum–positive patients had significantly higher cancer-specific mortality compared with F. nucleatum–negative patients [HR = 2.01; 95% confidence interval (CI), 1.22–3.23; P = 0.0068; Table 2]. In the multivariate Cox model adjusted for clinical, pathologic, and epidemiologic features, F. nucleatum positivity was associated with significantly higher cancer-specific mortality (multivariate HR = 1.78; 95% CI, 1.06–2.94; P = 0.032; Table 2). Similar results were observed for overall mortality.

Figure 2.

Kaplan–Meier curves for cancer-specific survival (A) and OS (B) in patients with esophageal cancer according to F. nucleatum DNA status in tumor tissues.

Figure 2.

Kaplan–Meier curves for cancer-specific survival (A) and OS (B) in patients with esophageal cancer according to F. nucleatum DNA status in tumor tissues.

Close modal
Table 2.

Cox regression analyses for cancer-specific survival

Univariate analysisMultivariate analysis
CharacteristicsHR (95% CI)PHR (95% CI)P
Age (for 10-year increase) 1.71 (0.50–6.10) 0.39   
Male(vs. female) 1.55 (0.73–4.02) 0.27   
Tobacco use (yes vs. no) 1.09 (0.60–2.20) 0.79   
Alcohol use (yes vs. no) 1.12 (0.60–2.33) 0.74   
Performance status 1–2 (vs. 0) 2.27 (1.36–3.67) 0.0020 2.16 (1.20–3.85) 0.011 
Comorbidity present (vs. absent) 1.09 (0.67–1.84) 0.72   
Upper tumor location (vs. lower) 1.05 (0.55–1.85) 0.87   
Preoperative therapy present (vs. absent) 3.58 (2.25–5.80) <0.0001   
Tumor stage III–IV (vs. stage I, II) 4.48 (2.75–7.54) <0.0001 2.83 (1.51–5.44) 0.0012 
Fusobacterium nucleatum positive (vs. negative) 2.01 (1.22–3.23) 0.0068 1.78 (1.06–2.94) 0.032 
Univariate analysisMultivariate analysis
CharacteristicsHR (95% CI)PHR (95% CI)P
Age (for 10-year increase) 1.71 (0.50–6.10) 0.39   
Male(vs. female) 1.55 (0.73–4.02) 0.27   
Tobacco use (yes vs. no) 1.09 (0.60–2.20) 0.79   
Alcohol use (yes vs. no) 1.12 (0.60–2.33) 0.74   
Performance status 1–2 (vs. 0) 2.27 (1.36–3.67) 0.0020 2.16 (1.20–3.85) 0.011 
Comorbidity present (vs. absent) 1.09 (0.67–1.84) 0.72   
Upper tumor location (vs. lower) 1.05 (0.55–1.85) 0.87   
Preoperative therapy present (vs. absent) 3.58 (2.25–5.80) <0.0001   
Tumor stage III–IV (vs. stage I, II) 4.48 (2.75–7.54) <0.0001 2.83 (1.51–5.44) 0.0012 
Fusobacterium nucleatum positive (vs. negative) 2.01 (1.22–3.23) 0.0068 1.78 (1.06–2.94) 0.032 

Survival analyses of interaction between F. nucleatum and other variables

We determined whether the influence of F. nucleatum on cancer-specific survival was modified by any of the clinical, pathologic, or epidemiologic variables evaluated. The effect of F. nucleatum was not significantly modified by age, year of surgery, performance status, tumor location, preoperative treatment, tumor size, or tumor stage (all P > 0.09; Fig. 3). Notably, we did not observe a modifying effect of the preoperative treatment on the relationship between F. nucleatum and cancer-specific survival rate (Pinteraction = 0.58).

Figure 3.

Relationship between F. nucleatum DNA status in esophageal cancer and cancer-specific survival. Loge (HRs) plots of cancer-specific survival rate in F. nucleatum DNA-positive and -negative groups are shown. PS, performance status.

Figure 3.

Relationship between F. nucleatum DNA status in esophageal cancer and cancer-specific survival. Loge (HRs) plots of cancer-specific survival rate in F. nucleatum DNA-positive and -negative groups are shown. PS, performance status.

Close modal

Tumor F. nucleatum DNA status and patient survival in esophageal SCC

SCC is the predominant type of esophageal cancer in the East, including Japan. We therefore also performed survival analyses, including only SCC (n = 300). F. nucleatum–positive patients with SCC had significantly lower cancer-specific survival compared with F. nucleatum–negative patients (log-rank P = 0.0012, univariate HR = 2.26; 95% CI, 1.34–3.72; P = 0.0026; multivariate HR = 1.98; 95% CI, 1.14–3.37; P = 0.016).

Upregulated pathways in F. nucleatum–positive esophageal cancer tissues

To clarify the mechanism whereby F. nucleatum may confer a poor prognosis, we performed enrichment analyses of KEGG pathways using the microarray data. The top 10 most enriched KEGG pathways associated with the significantly upregulated DEGs in F. nucleatum–positive esophageal cancer tissues are shown in Fig. 4A. Importantly, “cytokine–cytokine receptor interaction” was the top-ranked pathway (FDR < 0.001, fold enrichment > 1.95). A list of the chemokine DEGs (fold change > 2) in “cytokine–cytokine receptor interaction” is shown in Supplementary Table S2. We hypothesized that F. nucleatum might contribute to the acquisition of aggressive tumor behavior through the activation of chemokines. CCL20 was identified as the most upregulated chemokine (Supplementary Table S2), and we therefore evaluated the expression status of CCL20 in esophageal cancer tissues by IHC (Fig. 4B). We confirmed that the presence or absence of F. nucleatum was significantly associated with CCL20 expression status (Fig. 4C).

Figure 4.

F. nucleatum and chemokines in esophageal cancer. A, top 10 most enriched KEGG pathways among significantly upregulated DEGs in F. nucleatum–positive esophageal cancer tissues. Dashed lines, P values for the 10 top-ranked categories of KEGG pathways. The P values are expressed as the negative logarithm (base 10). B, immunostaining for CCL20 in esophageal cancer tissues. CCL20 positivity was observed in the cytoplasm and membrane of esophageal cancer cells. C, CCL20 expression scores were significantly higher in F. nucleatum–positive tissues (n = 20) compared with F. nucleatum–negative tissues (n = 20).

Figure 4.

F. nucleatum and chemokines in esophageal cancer. A, top 10 most enriched KEGG pathways among significantly upregulated DEGs in F. nucleatum–positive esophageal cancer tissues. Dashed lines, P values for the 10 top-ranked categories of KEGG pathways. The P values are expressed as the negative logarithm (base 10). B, immunostaining for CCL20 in esophageal cancer tissues. CCL20 positivity was observed in the cytoplasm and membrane of esophageal cancer cells. C, CCL20 expression scores were significantly higher in F. nucleatum–positive tissues (n = 20) compared with F. nucleatum–negative tissues (n = 20).

Close modal

We examined the prognostic impact of human microbiome F. nucleatum among 325 patients with resected esophageal cancers. It is becoming increasingly clear that the human microbiome influences cancer development and progression (5, 30–32). A better understanding of the mechanisms and contribution of the microbiota to human cancers may thus aid the development of novel approaches to cancer treatment and/or prevention. To the best of our knowledge, the current study provides the first evidence for the relationship between F. nucleatum and poor prognosis in esophageal cancer. In addition, using KEGG enrichment analysis, we demonstrated that F. nucleatum might contribute to the acquisition of aggressive tumor behavior through the activation of chemokines, such as CCL20.

Previous studies of the relationships between Fusobacterium species and clinical outcome in human cancers have been inconclusive (Supplementary Table S1). Two studies reported that tumor Fusobacterium species were associated with a poor prognosis in patients with colorectal cancer (32, 33). Mima and colleagues analyzed a molecular pathology epidemiologic database of more than 1,000 colorectal cancers and revealed that F. nucleatum DNA levels in colorectal cancer tissues were associated with shorter survival (24), while Flanagan and colleagues demonstrated a relationship between F. nucleatum and poor prognosis in 122 colorectal cancers (23). Regarding pancreatic cancer, Mitsuhashi and colleagues reported that tumor Fusobacterium status was independently associated with a poorer prognosis (33). Our current findings in relation to esophageal cancer are in agreement with these previous results. In contrast, however, two other studies (n = 99 and n = 511, respectively) of colorectal cancer found no associations between Fusobacterium detection and clinical outcome (20, 25). This discrepancy may be attributable to differences in patient cohorts or in the methods used to assess Fusobacterium species, or simply to chance variations between independent studies. However, our results demonstrated a clear association between increased F. nucleatum DNA content in esophageal cancer tissues and a poor prognosis, suggesting that F. nucleatum may be a suitable biomarker for identifying patients likely to experience inferior outcomes.

Experimental studies have provided mechanistic insights into the relationship between F. nucleatum and cancer progression. F. nucleatum is known to express the novel adhesin protein, FadA, on the bacterial cell surface (34). Rubinstein and colleagues revealed that FadA can bind to E-cadherin, activate β-catenin signaling, and promote colorectal cancer cell proliferation in in vitro and in vivo models (22). They also reported that colorectal cancer tissues showed elevated fadA gene levels, suggesting a potential role of FadA as a diagnostic and therapeutic target in human cancer. F. nucleatum was shown to inhibit T cell–mediated immune responses against colorectal tumors and promote tumor progression in the ApcMin/+ mouse model (35). Another study using colorectal cancer samples revealed an inverse association between tissue levels of F. nucleatum DNA and CD3+ T-cell density in tumor tissues (28). Collectively, these results suggest that F. nucleatum may exert immunosuppressive activity by inhibiting human T-cell responses. A third possible mechanism involves modulation of the tumor immune microenvironment. An in vivo study showed that colonization by F. nucleatum stimulated the secretion of immune cytokines, leading to colon tumorigenesis (35). Furthermore, our study revealed that the “cytokine–cytokine receptor interaction” was the most upregulated pathway in F. nucleatum–positive esophageal cancer, thereby supporting this third possible mechanism.

Accumulating evidence has demonstrated the crucial roles of chemokines and their receptors in tumor development and progression in several types of cancers (36–39). Kretschmer and colleagues recently reported that esophageal SCC cells modulated chemokine expression in fibroblasts, affecting the tumor immune response (40). The most upregulated chemokine in F. nucleatum–positive esophageal cancers in the current study was CCL20. An increasing number of studies have recently drawn attention to the roles of CCL20 and its physiologic sole receptor CCR6 in the development and progression of various type of cancers (41–43). An in vitro assay demonstrated that CCL20 stimulation promoted cancer cell proliferation and migration (44, 45,). In addition, CCL20 plays crucial roles in the migration of Treg lymphocytes (46, 47), and the accumulation of Treg lymphocytes is associated with shorter survival in human cancers (48, 49). Liu and colleagues recently reported that CCL20 was related to tumor infiltration of Treg lymphocytes in esophageal SCC, suggesting the importance of chemokines, such as CCL20, in immune surveillance in esophageal cancer patients (50). Further studies are needed to validate the current findings and to elucidate the mechanism(s) whereby F. nucleatum affects tumor behavior.

In conclusion, F. nucleatum was detected in esophageal cancer tissues and was associated with shorter survival, suggesting that it may serve as a useful prognostic biomarker. F. nucleatum might also contribute to the acquisition of aggressive tumor behavior through the activation of chemokines, such as CCL20.

No potential conflicts of interest were disclosed.

Conception and design: K. Yamamura, Y. Baba, K. Mima, K. Kinoshita, Y. Sakamoto, N. Yoshida

Development of methodology: K. Yamamura, Y. Baba, K. Mima, K. Miyake

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Y. Baba, K. Nakamura, N. Yoshida

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): K. Yamamura, Y. Baba, S. Nakagawa, K. Mima, K. Miyake

Writing, review, and/or revision of the manuscript: K. Yamamura, Y. Baba, Y. Sakamoto, Y. Yamashita, M. Watanabe, H. Baba

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): K. Yamamura, H. Sawayama, T. Ishimoto, M. Iwatsuki, Y. Sakamoto, Y. Yamashita, N. Yoshida, M. Watanabe, H. Baba

Study supervision: M. Iwatsuki, N. Yoshida, M. Watanabe, H. Baba

This work was supported in part by SGH Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Enzinger
PC
,
Mayer
RJ
. 
Esophageal cancer
.
N Engl J Med
2003
;
349
:
2241
52
.
2.
Kleinberg
L
,
Forastiere
AA
. 
Chemoradiation in the management of esophageal cancer
.
J Clin Oncol
2007
;
25
:
4110
7
.
3.
Brucher
BL
,
Swisher
SG
,
Konigsrainer
A
,
Zieker
D
,
Hartmann
J
,
Stein
H
, et al
Response to preoperative therapy in upper gastrointestinal cancers
.
Ann Surg Oncol
2009
;
16
:
878
86
.
4.
Wouters
MW
,
Karim-Kos
HE
,
le Cessie
S
,
Wijnhoven
BP
,
Stassen
LP
,
Steup
WH
, et al
Centralization of esophageal cancer surgery: does it improve clinical outcome?
Ann Surg Oncol
2009
;
16
:
1789
98
.
5.
Arthur
JC
,
Gharaibeh
RZ
,
Muhlbauer
M
,
Perez-Chanona
E
,
Uronis
JM
,
McCafferty
J
, et al
Microbial genomic analysis reveals the essential role of inflammation in bacteria-induced colorectal cancer
.
Nat Commun
2014
;
5
:
4724
.
6.
Louis
P
,
Hold
GL
,
Flint
HJ
. 
The gut microbiota, bacterial metabolites and colorectal cancer
.
Nat Rev Microbiol
2014
;
12
:
661
72
.
7.
Garrett
WS
. 
Cancer and the microbiota
.
Science
2015
;
348
:
80
6
.
8.
Ley
RE
,
Turnbaugh
PJ
,
Klein
S
,
Gordon
JI
. 
Microbial ecology: human gut microbes associated with obesity
.
Nature
2006
;
444
:
1022
3
.
9.
Furet
JP
,
Kong
LC
,
Tap
J
,
Poitou
C
,
Basdevant
A
,
Bouillot
JL
, et al
Differential adaptation of human gut microbiota to bariatric surgery-induced weight loss: links with metabolic and low-grade inflammation markers
.
Diabetes
2010
;
59
:
3049
57
.
10.
Ohkusa
T
,
Okayasu
I
,
Ogihara
T
,
Morita
K
,
Ogawa
M
,
Sato
N
. 
Induction of experimental ulcerative colitis by Fusobacterium varium isolated from colonic mucosa of patients with ulcerative colitis
.
Gut
2003
;
52
:
79
83
.
11.
Kostic
AD
,
Xavier
RJ
,
Gevers
D
. 
The microbiome in inflammatory bowel disease: current status and the future ahead
.
Gastroenterology
2014
;
146
:
1489
99
.
12.
Hotamisligil
GS
. 
Inflammation and metabolic disorders
.
Nature
2006
;
444
:
860
7
.
13.
Cani
PD
,
Osto
M
,
Geurts
L
,
Everard
A
. 
Involvement of gut microbiota in the development of low-grade inflammation and type 2 diabetes associated with obesity
.
Gut Microbes
2012
;
3
:
279
88
.
14.
Yoneda
M
,
Naka
S
,
Nakano
K
,
Wada
K
,
Endo
H
,
Mawatari
H
, et al
Involvement of a periodontal pathogen, Porphyromonas gingivalis on the pathogenesis of non-alcoholic fatty liver disease
.
BMC Gastroenterol
2012
;
12
:
16
.
15.
Furusho
H
,
Miyauchi
M
,
Hyogo
H
,
Inubushi
T
,
Ao
M
,
Ouhara
K
, et al
Dental infection of Porphyromonas gingivalis exacerbates high fat diet-induced steatohepatitis in mice
.
J Gastroenterol
2013
;
48
:
1259
70
.
16.
Cho
I
,
Blaser
MJ
. 
The human microbiome: at the interface of health and disease
.
Nat Rev Genet
2012
;
13
:
260
70
.
17.
Griffen
AL
,
Beall
CJ
,
Campbell
JH
,
Firestone
ND
,
Kumar
PS
,
Yang
ZK
, et al
Distinct and complex bacterial profiles in human periodontitis and health revealed by 16S pyrosequencing
.
Isme j
2012
;
6
:
1176
85
.
18.
Han
YW
. 
Fusobacterium nucleatum: a commensal-turned pathogen
.
Curr Opin Microbiol
2015
;
23
:
141
7
.
19.
Tahara
T
,
Shibata
T
,
Kawamura
T
,
Okubo
M
,
Ichikawa
Y
,
Sumi
K
, et al
Fusobacterium detected in colonic biopsy and clinicopathological features of ulcerative colitis in Japan
.
Dig Dis Sci
2015
;
60
:
205
10
.
20.
Castellarin
M
,
Warren
RL
,
Freeman
JD
,
Dreolini
L
,
Krzywinski
M
,
Strauss
J
, et al
Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma
.
Genome Res
2012
;
22
:
299
306
.
21.
Kostic
AD
,
Gevers
D
,
Pedamallu
CS
,
Michaud
M
,
Duke
F
,
Earl
AM
, et al
Genomic analysis identifies association of Fusobacterium with colorectal carcinoma
.
Genome Res
2012
;
22
:
292
8
.
22.
Rubinstein
MR
,
Wang
X
,
Liu
W
,
Hao
Y
,
Cai
G
,
Han
YW
. 
Fusobacterium nucleatum promotes colorectal carcinogenesis by modulating E-cadherin/beta-catenin signaling via its FadA adhesin
.
Cell Host Microbe
2013
;
14
:
195
206
.
23.
Flanagan
L
,
Schmid
J
,
Ebert
M
,
Soucek
P
,
Kunicka
T
,
Liska
V
, et al
Fusobacterium nucleatum associates with stages of colorectal neoplasia development, colorectal cancer and disease outcome
.
Eur J Clin Microbiol Infect Dis
2014
;
33
:
1381
90
.
24.
Mima
K
,
Nishihara
R
,
Qian
ZR
,
Cao
Y
,
Sukawa
Y
,
Nowak
JA
, et al
Fusobacterium nucleatum in colorectal carcinoma tissue and patient prognosis
.
Gut
. 
2015
Aug 26.
[Epub ahead of print]
.
25.
Ito
M
,
Kanno
S
,
Nosho
K
,
Sukawa
Y
,
Mitsuhashi
K
,
Kurihara
H
, et al
Association of Fusobacterium nucleatum with clinical and molecular features in colorectal serrated pathway
.
Int J Cancer
2015
;
137
:
1258
68
.
26.
Rice
TW
,
Blackstone
EH
,
Rusch
VW
. 
7th edition of the AJCC cancer staging manual: esophagus and esophagogastric junction
.
Ann Surg Oncol
2010
;
17
:
1721
4
.
27.
McShane
LM
,
Altman
DG
,
Sauerbrei
W
,
Taube
SE
,
Gion
M
,
Clark
GM
. 
Reporting recommendations for tumor marker prognostic studies (REMARK)
.
J Natl Cancer Inst
2005
;
97
:
1180
4
.
28.
Mima
K
,
Sukawa
Y
,
Nishihara
R
,
Qian
ZR
,
Yamauchi
M
,
Inamura
K
, et al
Fusobacterium nucleatum and T cells in colorectal carcinoma
.
JAMA Oncol
2015
;
1
:
653
61
.
29.
Huang da
W
,
Sherman
BT
,
Lempicki
RA
. 
Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources
.
Nat Protoc
2009
;
4
:
44
57
.
30.
Wu
S
,
Rhee
KJ
,
Albesiano
E
,
Rabizadeh
S
,
Wu
X
,
Yen
HR
, et al
A human colonic commensal promotes colon tumorigenesis via activation of T helper type 17 T cell responses
.
Nat Med
2009
;
15
:
1016
22
.
31.
Arthur
JC
,
Perez-Chanona
E
,
Muhlbauer
M
,
Tomkovich
S
,
Uronis
JM
,
Fan
TJ
, et al
Intestinal inflammation targets cancer-inducing activity of the microbiota
.
Science
2012
;
338
:
120
3
.
32.
Abreu
MT
,
Peek
RM
 Jr.
Gastrointestinal malignancy and the microbiome
.
Gastroenterology
2014
;
146
:
1534
46
.
33.
Mitsuhashi
K
,
Nosho
K
,
Sukawa
Y
,
Matsunaga
Y
,
Ito
M
,
Kurihara
H
, et al
Association of Fusobacterium species in pancreatic cancer tissues with molecular features and prognosis
.
Oncotarget
2015
;
6
:
7209
20
.
34.
Han
YW
,
Ikegami
A
,
Rajanna
C
,
Kawsar
HI
,
Zhou
Y
,
Li
M
, et al
Identification and characterization of a novel adhesin unique to oral fusobacteria
.
J Bacteriol
2005
;
187
:
5330
40
.
35.
Kostic
AD
,
Chun
E
,
Robertson
L
,
Glickman
JN
,
Gallini
CA
,
Michaud
M
, et al
Fusobacterium nucleatum potentiates intestinal tumorigenesis and modulates the tumor-immune microenvironment
.
Cell Host Microbe
2013
;
14
:
207
15
.
36.
Verbeke
H
,
Geboes
K
,
Van Damme
J
,
Struyf
S
. 
The role of CXC chemokines in the transition of chronic inflammation to esophageal and gastric cancer
.
Biochim Biophys Acta
2012
;
1825
:
117
29
.
37.
Tachezy
M
,
Zander
H
,
Gebauer
F
,
von Loga
K
,
Pantel
K
,
Izbicki
JR
, et al
CXCR7 expression in esophageal cancer
.
J Transl Med
2013
;
11
:
238
.
38.
Lukaszewicz-Zajac
M
,
Mroczko
B
,
Kozlowski
M
,
Szmitkowski
M
. 
The serum concentrations of chemokine CXCL12 and its specific receptor CXCR4 in patients with esophageal cancer
.
Dis Markers
2016
;
2016
:
7963895
.
39.
Ogura
M
,
Takeuchi
H
,
Kawakubo
H
,
Nishi
T
,
Fukuda
K
,
Nakamura
R
, et al
Clinical significance of CXCL-8/CXCR-2 network in esophageal squamous cell carcinoma
.
Surgery
2013
;
154
:
512
20
.
40.
Kretschmer
I
,
Freudenberger
T
,
Twarock
S
,
Yamaguchi
Y
,
Grandoch
M
,
Fischer
JW
. 
Esophageal squamous cell carcinoma cells modulate chemokine expression and hyaluronan synthesis in fibroblasts
.
J Biol Chem
2016
;
291
:
4091
106
.
41.
Nandi
B
,
Pai
C
,
Huang
Q
,
Prabhala
RH
,
Munshi
NC
,
Gold
JS
. 
CCR6, the sole receptor for the chemokine CCL20, promotes spontaneous intestinal tumorigenesis
.
PLoS One
2014
;
9
:
e97566
.
42.
Vicinus
B
,
Rubie
C
,
Stegmaier
N
,
Frick
VO
,
Kolsch
K
,
Kauffels
A
, et al
miR-21 and its target gene CCL20 are both highly overexpressed in the microenvironment of colorectal tumors: significance of their regulation
.
Oncol Rep
2013
;
30
:
1285
92
.
43.
Wang
GZ
,
Cheng
X
,
Li
XC
,
Liu
YQ
,
Wang
XQ
,
Shi
X
, et al
Tobacco smoke induces production of chemokine CCL20 to promote lung cancer
.
Cancer Lett
2015
;
363
:
60
70
.
44.
Campbell
AS
,
Albo
D
,
Kimsey
TF
,
White
SL
,
Wang
TN
. 
Macrophage inflammatory protein-3alpha promotes pancreatic cancer cell invasion
.
J Surg Res
2005
;
123
:
96
101
.
45.
Wang
B
,
Shi
L
,
Sun
X
,
Wang
L
,
Wang
X
,
Chen
C
. 
Production of CCL20 from lung cancer cells induces the cell migration and proliferation through PI3K pathway
.
J Cell Mol Med
2016
;
20
:
920
9
.
46.
Yamazaki
T
,
Yang
XO
,
Chung
Y
,
Fukunaga
A
,
Nurieva
R
,
Pappu
B
, et al
CCR6 regulates the migration of inflammatory and regulatory T cells
.
J Immunol
2008
;
181
:
8391
401
.
47.
Cook
KW
,
Letley
DP
,
Ingram
RJ
,
Staples
E
,
Skjoldmose
H
,
Atherton
JC
, et al
CCL20/CCR6-mediated migration of regulatory T cells to the Helicobacter pylori-infected human gastric mucosa
.
Gut
2014
;
63
:
1550
9
.
48.
Chen
KJ
,
Lin
SZ
,
Zhou
L
,
Xie
HY
,
Zhou
WH
,
Taki-Eldin
A
, et al
Selective recruitment of regulatory T cell through CCR6-CCL20 in hepatocellular carcinoma fosters tumor progression and predicts poor prognosis
.
PLoS One
2011
;
6
:
e24671
.
49.
Suzuki
H
,
Onishi
H
,
Morisaki
T
,
Tanaka
M
,
Katano
M
. 
Intratumoral FOXP3+VEGFR2+ regulatory T cells are predictive markers for recurrence and survival in patients with colorectal cancer
.
Clin Immunol
2013
;
146
:
26
33
.
50.
Liu
JY
,
Li
F
,
Wang
LP
,
Chen
XF
,
Wang
D
,
Cao
L
, et al
CTL- vs Treg lymphocyte-attracting chemokines, CCL4 and CCL20, are strong reciprocal predictive markers for survival of patients with oesophageal squamous cell carcinoma
.
Br J Cancer
2015
;
113
:
747
55
.