Combining fecal microbiome and metabolomics reveals diagnostic biomarkers for esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most predominant subtypes of esophageal cancer. The characteristics of the gut microbiome and its metabolites from patients with ESCC have not been adequately studied and discussed. In this study, 40 fecal samples (20 from ESCC patients and 20 from healthy controls) were analyzed by 16S rRNA gene sequencing and untargeted metabolomics. The data sets were analyzed individually and synthesized using various bioinformatics methods. Alpha and beta diversity indicated significant differences in microbial diversity and abundance between ESCC and healthy control feces. At the genus level, the abundance of Phascolarctobacterium, Sutterella, and Streptococcus was significantly increased in ESCC. At the genus level, linear discriminant analysis effect size identified two biomarkers: Bacteroides_stercoris and Prevotella_copri. Untargeted metabolomics analysis revealed 307 differential metabolites between ESCC and healthy control feces, with indoles and derivatives, tropane alkaloids, lipids, and lipid-like molecules in higher relative abundance in ESCC feces than in healthy control feces. Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that unsaturated fatty acids (FAs), ascorbate and aldarate metabolism, and hypoxia-inducible factor 1 signaling pathway were significantly associated with differential metabolite. Phenylethanolamine and despropionyl p-fluoro fentanyl could be used as reliable biomarkers to differentiate ESCC from healthy control. The correlation analysis showed that Prevotella may be involved in the synthesis of fatty acyl, carboxylic acids and derivatives, benzenes and substituted derivatives, organic oxygenates, and indoles and derivatives as metabolites. Fusicatenibacter and Lachnospira may be involved in the degradation of indoles and derivatives. Alistipes, Agathobacter, and Parabacteroides may be involved in the synthesis of indoles and derivatives with strong contributions. There is an intricate relationship between the gut microbiome and the levels of several metabolites (e.g., fatty acyls, carboxylic acids and derivatives, indoles, and derivatives). Microbial-associated metabolites can be used as diagnostic biomarkers in therapeutic exploration. Further analysis revealed that Prevotella, Alistipes, Agathobacter, and Parabacteroides might promote ESCC by regulating the synthesis of indoles and their derivatives. The results of this study provide favorable evidence for the early diagnosis of ESCC and subsequent individualized treatment and targeted interventions.IMPORTANCEWe describe for the first time the differences in fecal microbiome composition and metabolites between patients with esophageal squamous cell carcinoma (ESCC) and healthy controls by 16S rRNA gene sequencing and untargeted metabolomics. The results of this study provide a favorable basis for the early diagnosis of ESCC and subsequent targeted interventional therapy.

Characteristics and interplay of esophageal microbiota in esophageal squamous cell carcinoma.

BACKGROUND: Esophageal microbiota may influence esophageal squamous cell carcinoma (ESCC) pathobiology. Therefore, we investigated the characteristics and interplay of the esophageal microbiota in ESCC. METHODS: We performed 16S ribosomal RNA sequencing on paired esophageal tumor and tumor-adjacent samples obtained from 120 primarily ESCC patients. Analyses were performed using quantitative insights into microbial 2 (QIIME2) and phylogenetic investigation of communities by reconstruction of unobserved states 2 (PICRUSt2). Species found to be associated with ESCC were validated using quantitative PCR. RESULTS: The microbial diversity and composition of ESCC tumor tissues significantly differed from tumor-adjacent tissues; this variation between subjects beta diversity is mainly explained by regions and sampling seasons. A total of 56 taxa were detected with differential abundance between the two groups, such as R. mucilaginosa, P. endodontalis, N. subflava, H. Pylori, A. Parahaemolyticus, and A. Rhizosphaerae. Quantitative PCR confirmed the enrichment of the species P. endodontalis and the reduction of H. Pylori in tumor-adjacent tissues. Compared with tumor tissue, a denser and more complex association network was formed in tumor-adjacent tissue. The above differential taxa, such as H. Pylori, an unclassified species in the genera Sphingomonas, Haemophilus, Phyllobacterium, and Campylobacter, also participated in both co-occurrence networks but played quite different roles. Most of the differentially abundant taxa in tumor-adjacent tissues were negatively associated with the epidermal growth factor receptor (EGFR), erb-b2 receptor tyrosine kinase 2 (ERBB2), erb-b2 receptor tyrosine kinase 4 (ERBB4), and fibroblast growth factor receptor 1 (FGFR1) signaling pathways, and positively associated with the MET proto-oncogene, receptor tyrosine kinase (MET) and phosphatase and tensin homolog (PTEN) signaling pathways in tumors. CONCLUSION: Alterations in the microbial co-occurrence network and functional pathways in ESCC tissues may be involved in carcinogenesis and the maintenance of the local microenvironment for ESCC.

Fusobacterium nucleatum promotes esophageal squamous cell carcinoma progression via the NOD1/RIPK2/NF-κB pathway.

Fusobacterium nucleatum, found in the oral cavity, influences the progression of gastrointestinal cancers. Additionally, our previous results suggested that F. nucleatum is associated with poor patient prognosis in esophageal squamous cell carcinoma (ESCC). However, the mechanism by which F. nucleatum affects aggressive tumor behavior has yet to be elucidated. We have conducted this clinical, in vitro, and in vivo study to clarify the mechanism of ESCC progression induced by F. nucleatum. Transmission electron microscopy revealed that F. nucleatum invaded and occupied ESCC cells and impacted gene and protein expression. Comprehensive mRNA expression and pathway enrichment analyses of F. nucleatum-treated ESCC cells identified the "NF-κB" and "NOD-like receptor" signaling pathways as enriched. We confirmed the relationship between the presence of F. nucleatum and NF-κB activation in resected ESCC tissues. Furthermore, F. nucleatum-treated ESCC cells demonstrated enhanced growth ability, and NF-κB activation, as well as overexpression of NOD1 and phosphorylated RIPK2. Furthermore, treated cells showed accelerated tumor growth, with NF-κB activation in xenograft models. F. nucleatum invaded ESCC cells and induced the NF-κB pathway through the NOD1/RIPK2 pathway, leading to tumor progression.

Clinical impact of Fn-induced high expression of KIR2DL1 in CD8 T lymphocytes in oesophageal squamous cell carcinoma.

BACKGROUND: To analyze the correlation between the inducing effect of Fusobacterium nucleatum (Fn) on the surface expression of the inhibitory receptor KIR2DL1 on CD8+ T cells in oesophageal squamous cell carcinoma (ESCC) and the clinicopathological features and survival prognosis and to explore its clinical significance. METHODS: The inducing effect of Fn on CD8+ T cell surface inhibitory receptor KIR2DL1 expression was analyzed in a coculture system of human CD8+ T cells and ESCC cells infected with Fn. Fn infection and the expression of KIR2DL1 on CD8+ T cells were detected by RNAscope and immunohistochemistry in ESCC tissues, and the correlations between the inducing effect of Fn on KIR2DL1 expression on CD8+ T cells and clinicopathological features were analyzed. COX regression was used to analyze the influence of each factor on the prognosis of ESCC. Survival curves were plotted by the Kaplan-Meier method, and the effect of KIR2DL1 induction on survival time was analyzed by the log-rank test. RESULTS: In the coculture system, KIR2DL1 expression on the surface of CD8+ T cells increased with increasing Fn infection time. In ESCC tissues, Fn infection was significantly correlated with high KIR2DL1 expression on CD8+ T cells. The Fn + CD8+KIR2DL1 positive patients were predominantly males who were smokers and alcohol drinkers. Moreover, patients with Fn infection were characterized by poor tumour differentiation, advanced clinical stage, and a short survival time. Meanwhile, Fn + CD8+KIR2DL1 positive group was independent risk factor affecting the prognosis of ESCC patients. CONCLUSIONS: Long-term drinking and smoking lead to an extremely unhealthy oral environment in which Fn infection and colonization are more likely to occur, thus inducing high expression of KIR2DL1 on the surface of CD8+ T cells, which can weaken the antitumour immune response and promote the malignant progression of ESCC.HIGHLIGHTSFn induced high expression of KIR2DL1 CD8+ T cells in a time-dependent manner.Fn can reduce the response of tumour cells to CDDP.The inducing effect of Fn on CD8+ T cell surface KIR2DL1 expression was significantly associated with the poor prognosis of ESCC patients.

Associations of Porphyromonas gingivalis Infection and Low Beclin1 Expression With Clinicopathological Parameters and Survival of Esophageal Squamous Cell Carcinoma Patients.

Purpose: The present study focused on exploring the associations of Porphyromonas gingivalis (P. gingivalis) infection and low Beclin1 expression with clinicopathological parameters and survival of esophageal squamous cell carcinoma (ESCC) patients, so as to illustrate its clinical significance and prognostic value. Methods: Immunohistochemistry (IHC) was used to detect P. gingivalis infection status and Beclin1 expression in 370 ESCC patients. The chi-square test was adopted to illustrate the relationship between categorical variables, and Cohen's kappa coefficient was used for correlation analysis. Kaplan-Meier survival curves with the log-rank test were used to analyse the correlation of P. gingivalis infection and low Beclin1 expression with survival time. The effects of P. gingivalis infection and Beclin1 downregulation on the proliferation, migration and antiapoptotic abilities of ESCC cells in vitro were detected by Cell Counting Kit-8, wound healing and flow cytometry assays. For P. gingivalis infection of ESCC cells, cell culture medium was replaced with antibiotic-free medium when the density of ESCC cells was 70-80%, cells were inoculated with P. gingivalis at a multiplicity of infection (MOI) of 10. Result: P. gingivalis infection was negatively correlated with Beclin1 expression in ESCC tissues, and P. gingivalis infection and low Beclin1 expression were associated with differentiation status, tumor invasion depth, lymph node metastasis, clinical stage and prognosis in ESCC patients. In vitro experiments confirmed that P. gingivalis infection and Beclin1 downregulation potentiate the proliferation, migration and antiapoptotic abilities of ESCC cells (KYSE150 and KYSE30). Our results provide evidence that P. gingivalis infection and low Beclin1 expression were associated with the development and progression of ESCC. Conclusion: Long-term smoking and alcohol consumption causes poor oral and esophageal microenvironments and ESCC patients with these features were more susceptible to P. gingivalis infection and persistent colonization, and exhibited lower Beclin1 expression, worse prognosis and more advanced clinicopathological features. Our findings indicate that effectively eliminating P. gingivalis colonization and restoring Beclin1 expression in ESCC patients may contribute to preventation and targeted treatment, and yield new insights into the aetiological research on ESCC.

Characteristics of Oral Microbiota in Patients with Esophageal Cancer in China.

Gut microbiota dysbiosis is closely associated with intestinal carcinogenesis, but the oral microbiota of patients with esophageal squamous cell carcinoma who live in high-risk regions in China has not been fully characterized. In the current study, oral microbial diversity was investigated in 33 patients with esophageal squamous cell carcinoma and 35 healthy controls in Chongqing, China, by sequencing 16S rRNA of V3-V4 gene regions. There were statistically significant differences in oral microbiota between esophageal squamous cell carcinoma patients and controls as determined via unweighted pair-group analysis with arithmetic means. At the phylum level, in esophageal squamous cell carcinoma patients, there were comparatively greater amounts of Firmicutes (34.0% vs. 31.1%) and Bacteroidetes (25.3% vs. 24.9%) and lower amounts of Proteobacteria (17.0% vs. 20.1%). At the genus level, esophageal squamous cell carcinoma patients exhibited comparatively greater amounts of Streptococcus (17.3% vs. 14.5%) and Prevotella_7 (8.6% vs. 8.5%) and lower amounts of Neisseria (8.1% vs. 10.7%). Using a linear discriminant analysis effect size method, Planctomycetes and Verrucomicrobia were identified in the esophageal squamous cell carcinoma group. 10 genera were higher abundances identified in the healthy control group, and different 10 genera were identified in the esophageal squamous cell carcinoma group. In the present study, there were significant differences in oral microbial compositions of esophageal squamous cell carcinoma patients and healthy controls. Further longitudinal and mechanistic studies are needed to further characterize relationships between oral microbiota and esophageal squamous cell carcinoma.

Fusobacterium nucleatum predicts a high risk of metastasis for esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is the major type of esophageal cancer in China. The role of the bacteria present in ESCC tissue in neoplastic progression has not been fully elucidated. This study aimed to uncover different bacterial communities in ESCC tissues and examine the correlation between the abundance of the esophageal flora and clinicopathologic characteristics of ESCC. RESULTS: Microorganisms in tumors and normal tissues showed obvious clustering characteristics. The abundance of Fusobacterium (P = 0.0052) was increased in tumor tissues. The high level of Fusobacterium nucleatum was significantly associated with pT stage (P = 0.039) and clinical stage (P = 0.0039). The WES data showed that COL22A1, TRBV10-1, CSMD3, SCN7A and PSG11 were present in only the F. nucleatum-positive ESCC samples. GO and protein domain enrichment results suggested that epidermal growth factor might be involved in the regulation of cell apoptosis in F. nucleatum-positive ESCC. Both a higher mutational burden and F. nucleatum-positive was observed in tumors with metastasis than in tumors without metastasis. CONCLUSION: F. nucleatum is closely related to the pT stage and clinical stage of ESCC. The abundance of F. nucleatum and tumor mutation burden may be used in combination as a potential method to predict metastasis in ESCC.

Clinical Significance of Fusobacterium nucleatum Infection and Regulatory T Cell Enrichment in Esophageal Squamous Cell Carcinoma.

A variety of pathogenic microorganisms promote tumor occurrence and development through long-term colonization in the body. Fusobacterium nucleatum (F. nucleatum) is abundant in precancerous esophageal lesions and is closely related to the malignant progression of esophageal squamous cell carcinoma (ESCC). The invasion of exogenous microorganisms can reshape the immune microenvironment, make the immune system incapacitated, and assist tumor cells in immune escape. A variety of pathogenic microorganisms induce the recruitment of regulatory T cell (Tregs) to allow tumor cells to escape immune surveillance and provide favorable conditions for their own long-term colonization. Tregs are one of the major obstacles to tumor immunotherapy and have a significant positive correlation with the occurrence and development of many kinds of tumors. Because F. nucleatum can instantly enter cells and colonize for a long time, we speculated that F. nucleatum infection could facilitate the immune escape of tumor cells through enrichment of Tregs and promote the malignant progression of ESCC. In this study, we found a significant concordance between F. nucleatum infection and Tregs infiltration. Therefore, we propose the view that chronic infection of F. nucleatum may provide favorable conditions for long-term colonization of itself by recruiting Tregs and suppressing the immune response. At the same time, the massive enrichment of Treg may also weaken the immune response and assist in the long-term colonization of F. nucleatum. We analyzed the correlation between F. nucleatum infection with the clinicopathological characteristics and survival prognosis of the patients. F. nucleatum infection was found to be closely related to sex, smoking, drinking, degree of differentiation, depth of invasion, lymph node metastasis, and clinical stage. The degree of differentiation, depth of infiltration, lymph node metastasis, clinical stage, and F. nucleatum infection are independent risk factors affecting ESCC prognosis. Additionally, the survival rate and median survival time were significantly shortened in the F. nucleatum infection positive group. Therefore, we propose that long-term smoking and alcohol consumption cause poor oral and esophageal environments, thereby significantly increasing the risk of F. nucleatum infection. In turn, F. nucleatum infection and colonization may weaken the antitumor immune response through Treg enrichment and further assist in self-colonization, promoting the malignant progression of ESCC.

Porphyromonas gingivalis infection exacerbates oesophageal cancer and promotes resistance to neoadjuvant chemotherapy.

BACKGROUND: The effect of Porphyromonas gingivalis (Pg) infection on oesophageal squamous cell carcinoma (ESCC) prognosis, chemotherapeutic efficacy, and oesophageal cancer cell apoptosis resistance and proliferation remain poorly understood. METHODS: Clinicopathological data from 312 ESCC oesophagectomy patients, along with the computed tomography imaging results and longitudinal cancerous tissue samples from a patient subset (n = 85) who received neoadjuvant chemotherapy (NACT), were analysed. Comparison of overall survival and response rate to NACT between Pg-infected and Pg-uninfected patients was made by multivariate Cox analysis and Response Evaluation Criteria in Solid Tumours v.1.1 criteria. The influence of Pg on cell proliferation and drug-induced apoptosis was examined in ESCC patients and validated in vitro and in vivo. RESULTS: The 5-year overall survival was lower in Pg-positive patients, and infection was associated with multiple clinicopathological factors and pathologic tumour, node, metastasis stage. Of the 85 patients who received NACT, Pg infection was associated with a lower response rate and 5-year overall survival. Infection with Pg resulted in apoptosis resistance in ESCC and promoted ESCC cell viability, which was confirmed in longitudinal cancerous tissue samples. Pg-induced apoptosis resistance was dependent on fimbriae and STAT3. CONCLUSIONS: Pg infection is associated with a worse ESCC prognosis, reduced chemotherapy efficacy, and can potentiate the aggressive behaviour of ESCC cells.

Comparison of Oral and Esophageal Microbiota in Patients with Achalasia Before and After Peroral Endoscopic Myotomy.

BACKGROUND/AIMS: Patients with achalasia have a high incidence of esophageal squamous cell carcinoma (ESCC), which may be associated with alterations in oral and esophageal microbiota caused by food stasis. This study compared the oral and esophageal microbiota of patients with achalasia before and after peroral endoscopic myotomy (POEM). It also compared patients with achalasia to those with ESCC. MATERIALS AND METHODS: The study prospectively examined 6 patients with achalasia and 14 with superficial ESCC. Oral samples obtained from the buccal mucosa using a swab and esophageal samples obtained from the mid-esophagus using a brush via endoscopy were analyzed by 16S rRNA metagenome sequencing. Additionally, endoscopic and histological findings of patients with achalasia before and after POEM were prospectively compared. RESULTS: In patients with achalasia, Streptococcus was most abundant in both the oral and the esophageal microbiota, and these microbiota were significantly different. Although the overall structure of the oral and esophageal microbiota did not change after POEM, the relative abundance rate of Haemophilus and Neisseria increased in the esophagus, and endoscopic findings of inflammation improved after POEM (P = .04). The relative abundance of microbiota was not different among patients with achalasia from those with ESCC. CONCLUSIONS: The oral and esophageal microbiota were significantly different in patients with achalasia, and some of the composition of the esophageal microbiota changed after POEM. However, these findings and disease-specific microbiota should be further evaluated in large-scale studies.

Association between alcohol consumption and oesophageal microbiota in oesophageal squamous cell carcinoma.

BACKGROUND: Microbiota has been reported to play a role in cancer patients. Nevertheless, little is known about the association between alcohol consumption and resultant changes in the diversity and composition of oesophageal microbiota in oesophageal squamous cell carcinoma (ESCC). METHODS: We performed a hospital-based retrospective study of 120 patients with pathologically diagnosed primary ESCC. The relevant information for all study participants were collected through a detailed questionnaire. The differences in adjacent tissues between non-drinkers and drinkers were explored using 16S rRNA gene sequencing. Raw sequencing data were imported into QIIME 2 to analyse the diversity and abundance of microbiota. Linear discriminant analysis effect size (LEfSe) and unconditional logistic regression were performed to determine the bacterial taxa that were associated with drinking. RESULTS: The Shannon diversity index and Bray-Curtis distance of oesophageal microbiota were significantly different among drinkers(P < 0.05). The alcohol-related bacteria were primarily from the orders Clostridiales, Gemellales and Pasteurellales, family Clostridiaceae, Lanchnospiraceae, Helicobacteraceae, Alcaligenaceae, Bacteroidaceae, Pasteurellaceae and Gemellaceae; genus Clostridium, Helicobacter, Catonella, Bacteroides, Bacillus, Moraxella, and Bulleidia; and species B. moorei and longum (genus Bifidobacterium). In addition, the diversity and abundance of these microbiota were observed to be affected by the age, residential districts of the patients, and sampling seasons. Moreover, the higher the frequency and years of alcohol consumption, the lower was the relative abundance of genus Catonella that was observed. CONCLUSION: Alcohol consumption is associated with alterations in both the diversity and composition the of the oesophageal microbiota in ESCC patients.

Porphyromonas gingivalis promotes tumor progression in esophageal squamous cell carcinoma.

PURPOSE: Increasing evidence indicates that the microbiome may influence tumor growth and modulate the tumor microenvironment of gastrointestinal cancers. However, the role of oral bacteria in the development of esophageal squamous cell carcinoma (EsoSCC) has remained unclear. Herein, we investigated the relationship between the periodontal pathogen Porphyromonas gingivalis and EsoSCC. METHODS: To identify bacterial biomarkers associated with EsoSCC, we analyzed microbiomes in oral biofilms. The presence of P. gingivalis in esophageal tissues and relationships of P. gingivalis infection with clinicopathologic characteristics in 156 patients with EsoSCC were assessed using immunohistochemistry. The role of P. gingivalis infection in in vitro and in vivo EsoSCC progression was also assessed. RESULTS: Microbiota profiles in oral biofilms revealed that P. gingivalis abundance was associated with an increased risk of EsoSCC development. In total, 57% of patients with EsoSCC were found to be infected with P. gingivalis. The presence of P. gingivalis was found to be associated with advanced clinical stages and a poor prognosis. It was also found to be associated with an elevated esophageal cancer incidence in a 4-nitroquinoline 1-oxide-induced mouse model and with an increased xenograft tumor growth. P. gingivalis infection increased interleukin (IL)-6 production and it promoted epithelial-mesenchymal transition and the recruitment of myeloid-derived suppressor cells. Furthermore, inhibited IL-6 signaling attenuated the tumor-promoting effects of P. gingivalis in 4-nitroquinoline 1-oxide-treated mice and xenograft mouse models. CONCLUSIONS: Our data indicate that P. gingivalis may promote esophageal cancer development and progression. Direct targeting of P. gingivalis or concomitant IL-6 signaling may be a promising strategy to prevent and/or treat EsoSCC associated with P. gingivalis infection.

The Upper Digestive Tract Microbiome and Oesophageal Squamous Cell Carcinoma: Epidemiology, Pathogenesis, and Clinical Implications in Africa.

The study of the microbiome has significantly contributed to our understanding of complex diseases including cancer, with a profound influence of the microbiota on clinical prognosis and the efficacy of cancer treatments. Oesophageal cancer is positioned amongst the most aggressive malignant diseases, resulting from a complex interaction between anthropometric, genetic, immune response, and environmental factors. Oesophageal squamous cell carcinoma (OSCC) is the most common type of oesophageal cancer and is a serious burden in Eastern Africa, in the area known as the African oesophageal cancer corridor (AOCC). OSCC is often diagnosed at a late stage, with patients already suffering from severe malnutrition and dehydration due to swallowing difficulties, leading to high mortality rates. So far, aetiological factors have been individually analysed with an inappropriate contextualisation. The upper digestive tract microbiome has been proposed to contribute to the onset and progression of OSCC but with limited understanding of the mechanisms behind this interaction. Data on African populations are limited, and the aetiology of AOCC is still poorly understood. This review discusses the current knowledge of the aetiology of OSCC in Africa, with special focus on the probable influence of the upper digestive tract microbiota.

Fusobacterium nucleatum confers chemoresistance by modulating autophagy in oesophageal squamous cell carcinoma.

BACKGROUND: Fusobacterium nucleatum (F. nucleatum) is a gut microbe implicated in gastrointestinal tumorigenesis. Predicting the chemotherapeutic response is critical to developing personalised therapeutic strategies for oesophageal cancer patients. The present study investigated the relationship between F. nucleatum and chemotherapeutic resistance in oesophageal squamous cell carcinoma (ESCC). METHODS: We examined the relationship between F. nucleatum and chemotherapy response in 120 ESCC resected specimens and 30 pre-treatment biopsy specimens. In vitro studies using ESCC cell lines and co-culture assays further uncovered the mechanism underlying chemotherapeutic resistance. RESULTS: ESCC patients with F. nucleatum infection displayed lesser chemotherapeutic response. The infiltration and subsistence of F. nucleatum in the ESCC cells were observed by transmission electron microscopy and laser scanning confocal microscopy. We also observed that F. nucleatum modulates the endogenous LC3 and ATG7 expression, as well as autophagosome formation to induce chemoresistance against 5-FU, CDDP, and Docetaxel. ATG7 knockdown resulted in reversal of F. nucleatum-induced chemoresistance. In addition, immunohistochemical studies confirmed the correlation between F. nucleatum infection and ATG7 expression in 284 ESCC specimens. CONCLUSIONS: F. nucleatum confers chemoresistance to ESCC cells by modulating autophagy. These findings suggest that targeting F. nucleatum, during chemotherapy, could result in variable therapeutic outcomes for ESCC patients.

Porphyromonas gingivalis promotes progression of esophageal squamous cell cancer via TGFß-dependent Smad/YAP/TAZ signaling.

Microbial dysbiosis in the upper digestive tract is linked to an increased risk of esophageal squamous cell carcinoma (ESCC). Overabundance of Porphyromonas gingivalis is associated with shorter survival of ESCC patients. We investigated the molecular mechanisms driving aggressive progression of ESCC by P. gingivalis. Intracellular invasion of P. gingivalis potentiated proliferation, migration, invasion, and metastasis abilities of ESCC cells via transforming growth factor-ß (TGFß)-dependent Drosophila mothers against decapentaplegic homologs (Smads)/Yes-associated protein (YAP)/Transcriptional coactivator with PDZ-binding motif (TAZ) activation. Smads/YAP/TAZ/TEA domain transcription factor1 (TEAD1) complex formation was essential to initiate downstream target gene expression, inducing an epithelial-mesenchymal transition (EMT) and stemness features. Furthermore, P. gingivalis augmented secretion and bioactivity of TGFß through glycoprotein A repetitions predominant (GARP) up-regulation. Accordingly, disruption of either the GARP/TGFß axis or its activated Smads/YAP/TAZ complex abrogated the tumor-promoting role of P. gingivalis. P. gingivalis signature genes based on its activated effector molecules can efficiently distinguish ESCC patients into low- and high-risk groups. Targeting P. gingivalis or its activated effectors may provide novel insights into clinical management of ESCC.

Porphyromonas gingivalis Promotes the Proliferation and Migration of Esophageal Squamous Cell Carcinoma through the miR-194/GRHL3/PTEN/Akt Axis.

Recent studies have revealed that Porphyromonas gingivalis is closely related to the occurrence and progression of esophageal squamous cell carcinoma (ESCC). However, the underlying mechanism of P. gingivalis in ESCC has not been well elucidated. To explore the mechanism of P. gingivalis infection in ESCC, cellular proliferation, invasion, and migration models of KYSE-30 and KYSE-150 cells infected by P. gingivalis at a multiplicity of infection (MOI) of 10 were established. The results showed that P. gingivalis infection could drastically increase the proliferation, invasion, and migration ability of ESCC. Furthermore, the results of high-throughput sequencing showed that miR-194 was considerably upregulated in infected cells compared with control cells, which was further verified by qRT-PCR. The inhibition or overexpression of miR-194 had a significant effect on KYSE-30 and KYSE-150 cell migration and invasion. Additionally, the levels of GRHL3 and PTEN were decreased in P. gingivalis-infected esophageal cancer cells compared with uninfected esophageal cancer cells. Furthermore, dual-luciferase experiments confirmed that GRHL3 is a direct target of miR-194. In addition, the GRHL3-related pathway was investigated, and the levels of GRHL3 and PTEN were downregulated while the level of p-Akt was upregulated after P. gingivalis infection. Taken together, these findings indicated that P. gingivalis might promote ESCC proliferation and migration via the miR-194/GRHL3/PTEN/Akt signaling axis.

Oral Microbiome in Patients with Oesophageal Squamous Cell Carcinoma.

To investigate the oral microflora of patients with oesophageal squamous cell carcinoma (ESCC), saliva samples were collected from 20 patients with ESCC and 21 healthy controls. The V3-V4 region of 16S rDNA was amplified and sequenced by the Illumina MiSeq high-throughput sequencing platform. The final sequences were used for OTU analysis. Alpha and beta diversity analysis showed that the bacterial diversity and richness of the ESCC group were lower than those of the control group, while the variability of the ESCC group was higher than that of the control group. According to the Metastats difference analysis and LEfSe analysis, the high risk of ESCC may be related to Actinomyces and Atopobium, while the healthy control group is closely related to Fusobacterium and Porphyromonas (the analysis was performed at the genus level). The establishment of the relationship between oral microbiota and risk of ESCC may lead to significant advances in understanding the aetiology of cancer and may open a new research paradigm for cancer prevention.

[Gut Microbiome Differences between Gastrointestinal Cancer Patients and Healthy People].

Objective To compare the differences in fecal flora among patients with esophageal cancer,gastric cancer,or colorectal cancer and between patients with gastrointestinal tumors and healthy people.Methods The 16S rRNA method was used to analyze the differences in fecal flora among 13 patients with esophageal squamous cell carcinoma,23 patients with gastric cancer,6 patients with colorectal cancer,and 49 healthy persons.Results Bifidobacterium,Faecalibacterium prausnitzii,and Ruminococcus callidus were less abundant in the fecal flora of cancer patients than in those of healthy controls(all P<0.05).Some species of Firmicutes and Actinobacteria were significantly reduced in the feces of patients with esophageal cancer or gastric cancer than in healthy people(P<0.05),whereas others showed consistency with the intestinal cancer group.Anti-tumor treatment,antibiotics,and lactic acid could affect the fecal flora of cancer patients.Conclusion The gut microbiota compositions(mainly Firmicutes and Actinobacteria)and some specific bacteria species in the feces of patients with esophageal cancer and gastric cancer are similar to those in the feces of patients with intestinal cancer,suggesting these bacteria may be involved in the development of upper gastrointestinal tumors.

A Comparison of Biopsy and Mucosal Swab Specimens for Examining the Microbiota of Upper Gastrointestinal Carcinoma.

BACKGROUND: There is currently no optimal sampling method for upper gastrointestinal (UGI) tract microbiota. We compared biopsies and mucosal swab specimens for microbial sampling from patients with UGI carcinoma. METHODS: A total of 67 patients with esophageal squamous cell carcinoma (ESCC) and 36 patients with gastric cardia adenocarcinoma (GCA) were recruited in the Linxian Cancer Hospital (Henan, China). Sterile biopsies and swabs were used to collect paired samples from the resection specimens from carcinoma and adjacent normal tissue. Data from 16S rRNA gene sequencing were processed using QIIME2 to evaluate differences in alpha and beta diversity and taxonomic relative abundances between specimen types. RESULTS: Alpha diversity was not significantly different between swab specimens and biopsies, both for ESCC and GCA. Paired specimens were correlated for both sample types from ESCC (ρ > 0.6, P < 0.001) but not GCA (ρ < 0.4, P > 0.05). For beta diversity, distinct clustering by sampling method was not observed for adjacent normal or tumor tissue from ESCC or GCA. There was a high correlation for weighted UniFrac and Bray-Curtis distance only in ESCC paired specimens (ρ > 0.6, P = 0.001). The 10 dominant bacterial genera were similar between swab and biopsy specimens. However, higher levels of Veillonella (P = 0.0002) and Streptococcus (P = 0.0002) were detected in ESCC adjacent normal and GCA carcinoma swabs, respectively, compared with the biopsies. CONCLUSIONS: Mucosal swab specimens and biopsies could yield similar microbial profiles from ESCC but not GCA. Both can be used to characterize UGI microbiota; one sampling method should be selected for future studies. IMPACT: This study provides insight for planning microbiota collections from the UGI tract.

Intratumoral Fusobacterium Nucleatum Levels Predict Therapeutic Response to Neoadjuvant Chemotherapy in Esophageal Squamous Cell Carcinoma.

PURPOSE: Emerging evidence indicates that gut microbiome plays a crucial role in the cancer pathogenesis. Although Fusobacterium nucleatum (F. nucleatum) is associated with poor prognosis in multiple cancers, its clinical significance in predicting response to chemotherapy in patients with esophageal squamous cell carcinoma (ESCC) remains unclear. EXPERIMENTAL DESIGN: The F. nucleatum levels were quantified by qPCR assays in tumor tissues from 551 patients with ESCC from two independent cohorts, including 101 patients who received neoadjuvant chemotherapy prior to curative resection. Associations between F. nucleatum burden and recurrence-free survival (RFS), as well with chemotherapeutic response were evaluated using response evaluation criteria in solid tumors (RECISTs), primary tumor metabolic response defined by maximum standardized uptake value (SUVmax) changes in positron emission tomography-CT (PET/CT), and pathologic tumor regression grade (TRG). RESULTS: High burden of F. nucleatum in patients with ESCC associated with poor RFS in both training [log-rank P = 0.02; HR = 1.61; P = 0.03] and validation cohorts (log-rank P = 0.003; HR = 1.96; P = 0.004). Importantly, patients with ESCC with high levels of F. nucleatum displayed poor chemotherapeutic response for all three evaluation methods: RECIST (P = 0.04), SUVmax change in PET/CT (P = 0.0004), and TRG (P = 0.003). CONCLUSIONS: We conclude that high levels of intratumoral F. nucleatum have a prognostic significance for predicting poor RFS in patients with ESCC. More importantly, our data indicates that higher F. nucleatum burden correlates with poor response to neoadjuvant chemotherapy, suggesting the possibility that an antibiotic intervention against this bacterium may significantly improve therapeutic response in patients with ESCC.