Integrative methylome and transcriptome analysis reveals epigenetic regulation of Fusobacterium nucleatum in laryngeal cancer.

The aetiological mechanisms of Fusobacterium nucleatum in laryngeal cancer remain unclear. This study aimed to reveal the epigenetic signature induced by F. nucleatum in laryngeal squamous cell carcinoma (LSCC). Combined analysis of methylome and transcriptome data was performed to address the functional role of F. nucleatum in laryngeal cancer. Twenty-nine differentially expressed methylation-driven genes were identified by mapping the methylation levels of significant differential methylation sites to the expression levels of related genes. The combined analysis revealed that F. nucleatum promoted Janus kinase 3 (JAK3) gene expression in LSCC. Further validation found decreased methylation and elevated expression of JAK3 in the F. nucleatum-treated LSCC cell group; F. nucleatum abundance and JAK3 gene expression had a positive correlation in tumour tissues. This analysis provides a novel understanding of the impact of F. nucleatum in the methylome and transcriptome of laryngeal cancer. Identification of these epigenetic regulatory mechanisms opens up new avenues for mechanistic studies to explore novel therapeutic strategies.

Metagenomic shotgun sequencing reveals the enrichment of Salmonella and Mycobacterium in larynx due to prolonged ethanol exposure.

The exposure of ethanol increases the risk of head and neck inflammation and tumor progression. However, limited studies have investigated the composition and functionality of laryngeal microbiota under ethanol exposure. We established an ethanol-exposed mouse model to investigate the changes in composition and function of laryngeal microbiota using Metagenomic shotgun sequencing. In the middle and late stages of the experiment, the laryngeal microbiota of mice exposed to ethanol exhibited obvious distinguished from that of the control group on principal-coordinate analysis (PCoA) plots. Among the highly abundant species, Salmonella enterica and Mycobacterium marinum were likely to be most impacted. Our findings indicated that the exposure to ethanol significantly increased their abundance in larynxes in mice of the same age, which has been confirmed through FISH experiments. Among the species-related functions and genes, metabolism is most severely affected by ethanol. The difference was most obvious in the second month of the experiment, which may be alleviated later because the animal established tolerance. Notable enrichments concerning energy, amino acid, and carbohydrate metabolic pathways occurred during the second month under ethanol exposure. Finally, based on the correlation between species and functional variations, a network was established to investigate relationships among microbiota, functional pathways, and related genes affected by ethanol. Our data first demonstrated the continuous changes of abundance, function and their interrelationship of laryngeal microbiota under ethanol exposure by Metagenomic shotgun sequencing. Importance: Ethanol may participate in the inflammation and tumor progression by affecting the composition of the laryngeal microbiota. Here, we applied the metagenomic shotgun sequencing instead of 16 S rRNA sequencing method to identify the laryngeal microbiota under ethanol exposure. Salmonella enterica and Mycobacterium marinum are two dominant species that may play a role in the reconstruction of the laryngeal microenvironment, as their local abundance increases following exposure to ethanol. The metabolic function is most evidently impacted, and several potential metabolic pathways could be associated with alterations in microbiota composition. These findings could help us better understand the impact of prolonged ethanol exposure on the microbial composition and functionality in the larynx.

Fusobacterium nucleatum facilitates proliferation and autophagy by activating miR-361-3p/NUDT1 axis through oxidative stress in hypopharyngeal squamous cell carcinoma.

BACKGROUND: To investigate how Fusobacterium nucleatum (Fn) promotes oxidative stress and mediates proliferation and autophagy in hypopharyngeal squamous cell carcinoma (HPSCC). METHODS: The prognosis for 82 HPSCC cases was retrospectively analyzed. HPSCC cell line FaDu was co-cultured with Fn. Knockdown of NUDT1 (shNUDT1 group) was done after observing DNA damage response. CCK8 and tumorigenesis assays for proliferation observation, mitochondria ROS (MitoROS) measurement to examine intracellular oxidative stress, and ELISA to analyze concentration of 8-oxo-2'-deoxyguanosine (8-oxo-dG) in cells. Dual-luciferase reporter assays clarified miR-361-3p connection with NUDT1. Autophagy flow was observed using electron microscopy and related proteins. RESULTS: Fn was highly associated with NUDT1. The shNUDT1 group experienced lower proliferation compared with normal FaDu (NC group) in vivo and in vitro. The shNUDT1 group showed 8-oxo-dG and γH2AX to be elevated. Intracellular ROS decreased in shNUDT1Fn group when compared to Fn group. Upregulating miR-361-3p could suppress NUDT1 expression and downstream proliferation and autophagy. Fn modulated miR-361-3p via OH-, which could be proven by H2O2 assay and N-acetylcysteine. CONCLUSIONS: Higher Fn in HPSCC patients suggests poorer prognosis. NUDT1 might affect cell proliferation and autophagy and modulate DNA damage response. The oxidative stress induced miR-361-3p/NUDT1 axis is first introduced in microbiome-carcinoma research.

Tumour microbiota structure predicts hypopharyngeal carcinoma recurrence and metastasis.

Objectives: The relationship between microbiota and HPSCC recurrence and metastasis remains uncertain. This study aimed to investigate the role of the tumour microbiota in the disease-free survival (DFS) of HPSCC patients. Materials and methods: Formalin-fixed paraffin-embedded (FFPE) tumour tissues were collected from 103 patients with HPSCC for 16S rRNA sequencing. We analysed the tumour microbiota in HPSCC patients with recurrence/metastasis and nonrecurrence/metastasis. The linear predictor score (LPS) was calculated based on the Cox regression model to assess the risk of recurrence and metastasis. Then, a time-dependent ROC curve was used to evaluate the prognostic power of the LPS. Results: The phyla Bacteroidota, Firmicutes and Proteobacteria were the most abundant bacterial taxa in the tumour tissues. Eubacterium_coprostanoligenes_group (hazard ratio [HR] = 0.289, 95% confidence interval [CI] 0.137-0.608, p= 0.001) and Prevotella (HR = 3.744, 95% CI 1.439-9.738, p= 0.007) were independent predictors of DFS. The predicting classifier for recurrence and metastasis risk yielded an area under the curve (AUC) of 0.838 at 3 years and 0.860 at 5 years. Conclusion: Our study demonstrated the relationship between tumour microbiota and recurrence and metastasis in patients with HPSCC.

Fusobacterium nucleatum impairs DNA mismatch repair and stability in patients with squamous cell carcinoma of the head and neck.

BACKGROUND: Dysbiosis of the laryngeal microbiota has been demonstrated to the development of head and neck squamous cell carcinoma (HNSCC), but the association of Fusobacterium and Fusobacterium nucleatum (F. nucleatum) with DNA mismatch repair (MMR) and microsatellite instability (MSI) has not been investigated. METHODS: The abundance of Fusobacterium and F. nucleatum, the status of deficient MMR (dMMR) and MSI, and MMR-related gene expression were analyzed in 171 HNSCC tissues, 61 paired para-tumor tissues, and 60 vocal cord polyp tissues. The molecular mechanism of F. nucleatum and MMR-related gene expression were investigated in two human HNSCC cell lines (Tu 686 and FD-LSC-1). RESULTS: Our results demonstrated that a high Fusobacterium abundance was detected in the HNSCC tissues and was exaggerated in the recurrent patients. We further found that a high Fusobacterium abundance was detected in the HNSCC tissues with dMMR and MSI. The Fusobacterium abundance was negatively correlated with the expression of MLH1, MSH2, and MSH6 in the HNSCC tissues. The Fusobacterium abundance was closely associated with the F. nucleatum abundance in the HNSCC tissues. F. nucleatum increased miR-205-5p expression to suppress MLH1, MSH2, and MSH6 expression via the TLR4- and MYD88-dependent innate immune signaling pathway, resulting in dMMR, DNA damage, and cell proliferation in HNSCC. CONCLUSIONS: F. nucleatum impacts HNSCC epigenetic changes in tissues with dMMR to promote DNA damage and cell proliferation by suppressing MMR-related gene expression via the TLR4/MYD88/miR-205-5p signaling pathway, which is valuable in the development of efficient strategies for HNSCC prevention and treatment. LAY SUMMARY: This study clearly indicates that Fusobacterium induced head and neck squamous cell carcinoma (HNSCC) aggressiveness to affect poor prognosis in HNSCC patients by epigenetic alteration of DNA mismatch repair (MMR) and microsatellite instability. Moreover, the research has shown that Fusobacterium nucleatum ( F. nucleatum ) impacts HNSCC epigenetic changes in tissues with deficient MMR to promote DNA damage and cell proliferation by suppressing MMRrelated gene expression via the TLR4/MYD88/miR-205-5p signaling pathway.

Dual inhibition of autophagy and PI3K/mTOR pathway as a potential therapeutic strategy against laryngeal squamous cell carcinoma.

Background: New and effective chemotherapy or targeted therapy strategies are needed against laryngeal squamous cell carcinoma (LSCC). We aimed to explore the antitumor effect of dual PI3K/mTOR inhibitor combined with autophagy suppression on LSCC and its underlying mechanism. Methods: Hep-2 and AMC-HN-8 cell lines were treated with the Akt inhibitor LY294002, mTOR inhibitor rapamycin, and dual inhibitor NVP-BEZ235 separately. The biological characteristics of in vitro proliferation, cell cycle, apoptosis, migration, invasion, and autophagy were analyzed, and the expression levels of PI3K/Akt/mTOR pathway-related proteins were also measured. The in vivo effects of NVP-BEZ235 combined with inhibition of autophagy using pharmacological inhibitor was further assessed. Results: Compared with Akt or mTOR inhibitor, NVP-BEZ235 had the most significant biological effects on LSCC cells. When combined with various autophagy inhibitors, along with siRNA against ATG7, NVP-BEZ235 showed a synergic antitumor effect in LSCC through increasing cell apoptosis and death both in vitro and vivo. Conclusions: NVP-BEZ235 exerted potent antitumor effects on LSCC, especially when combined with the autophagy inhibitor both in vitro and vivo, providing convincing experimental data for new molecular targeted therapy for LSCC.

The Prognostic Capacity of Systemic Inflammation Response Index, Neutrophil-to-Lymphocyte Ratio, Lymphocyte-to-Monocyte Ratio, and Platelet-to-Lymphocyte Ratio in Patients with Hypopharyngeal Squamous Cell Carcinoma.

INTRODUCTION: Systemic inflammation response index (SIRI), neutrophil-to-lymphocyte ratio (NLR), and platelet-to-lymphocyte ratio (PLR) have been proposed as peripheral blood biomarkers. We compared these blood biomarkers to identify the best predictor in patients with hypopharyngeal squamous cell carcinoma (HPSCC). METHODS: We conducted a retrospective study on 304 patients with HPSCC. SIRI was divided into three groups using X-tile version 3.6.1. The optimal cut-off points for NLR, LMR, and PLR were selected through RStudio. We compared the prognostic capacity of SIRI with that of NLR, LMR, and PLR using receiver operating characteristic curves. RESULTS: Smoking, cancer in the postcricoid region, lymph node metastasis (N+), extracapsular invasion, SIRI in the highest tertile (>2.80), and LMR in the lowest tertile (<5.0) may cause poor 5-year overall survival (OS) in patients with HPSCC. Local and distant recurrences may occur earlier in those with lymph node metastasis and a tumor invading beyond the mucosa layer. CONCLUSIONS: SIRI was a better predictor of OS than LMR, PLR, and NLR in HPSCC patients. SIRI in the highest tertile (>2.80) and LMR in the lowest tertile (<5.0) may cause poor 5-year OS.

Cross-comparison of microbiota in the oropharynx, hypopharyngeal squamous cell carcinoma and their adjacent tissues through quantitative microbiome profiling.

Aims: To clarify the absolute abundance of microbial communities on hypopharyngeal squamous cell carcinoma and their correlation to those in the oropharynx. Methods: Clinical data, swabs, and tissue samples from 27 HPSCC patients were collected in this study and divided into three sampling groups: 19 oropharyngeal mucosa (OPM), 27 hypopharyngeal carcinomas tissues (HC), and 26 corresponding adjacent tissues (AT). Relative microbiome profiling (RMP), and quantitative microbiome profiling (QMP) of 16S rRNA amplicon sequencing were used for analysis. Results: Beta-diversity showed that abundance and phylogenetic tree in OPM group were less when compared to either HC and AT. Although HC and AT were found to have similar microbiota, Bray-Curtis based beta-diversity still highlighted differences. Fusobacterium, Porphyromonas, Haemophilus, and Peptostreptococcus at the genus level in OPM were positively correlated with HC. After categorizing HC through TNM staging, the abundance of genera Fusobacterium, Parvimonas, and Dialister were found to be enhanced in higher T classifications (T3-4) and advanced stages (Ⅳ). Conclusions: QMP yielded more comprehensive results than RMP. Dysbiosis was found in OPM groups and could be used to narrow down differential microbiome for the HC group. Genera of Parvimonas, Fusobacterium, and Dialister were deemed asrisk factors of advanced HPSCC.

A positive feed-forward loop between Fusobacterium nucleatum and ethanol metabolism reprogramming drives laryngeal cancer progression and metastasis.

Alcohol consumption, which affects the structure and composition of the laryngeal microbiota, is one of the most important risk factors for laryngeal squamous cell cancer (LSCC). Our results demonstrated that high enrichment of Fusobacterium nucleatum (F. nucleatum) in LSCC was associated with poor prognosis. F. nucleatum increased miR-155-5p and miR-205-5p expression to suppress alcohol dehydrogenase 1B (ADH1B) and transforming growth factor ß receptor 2 (TGFBR2) expression by activating innate immune signaling, resulting in ethanol metabolism reprogramming to allow F. nucleatum accumulation and PI3K/AKT signaling pathway activation to promote epithelial-mesenchymal transition, further exacerbating the uncontrolled progression and metastasis of LSCC. Therefore, the positive feed-forward loop between F. nucleatum and ethanol metabolism reprogramming promotes cell proliferation, migration, and invasion to affect LSCC patient prognosis. The amount of F. nucleatum is a potential prognostic biomarker, which yields valuable insight into clinical management that may improve the oncologic outcome of patients with LSCC.

Association Between Alcohol Dehydrogenase Polymorphisms and the Recurrence of Laryngeal Carcinoma.

OBJECTIVES: Alcohol consumption is closely associated with prognosis for laryngeal squamous cell carcinoma (LSCC) patients. As key enzymes in ethanol metabolism, proteins in the alcohol dehydrogenase (ADH) family make for valuable targets to establish a novel predictive nomogram model. This study attempts to do so by focusing on the single nucleotide polymorphisms (SNPs) of ADH1B and ADH1C in LSCC. METHODS: Sixty eight LSCC patients that were followed up for more than 10 years were retrospectively analyzed. Endpoints of the current study included disease-free survival and overall survival. Survival analyses were performed using the Kaplan-Meier method and evaluated by log-rank test. The prognostic value of eight ADH1B SNPs and three ADH1C SNPs were evaluated using univariate and multivariate Cox regression analyses. A nomogram model for disease-free survival was established and evaluated using the receiver operating characteristic curve, the C-index, and a calibration plot. RESULTS: Significant association was exhibited between rs17033 (p < 0.001) and rs1229984 (p = 0.002) with an increase in LSCC recurrence rate on Kaplan-Meier curves. Multivariate logistic regression analysis revealed that the rs17033 polymorphism of ADH1B was independently associated with an increased risk of LSCC recurrence (HR = 3.325, 95% CI = 1.684-6.566, p = 0.001). Based on these findings, a prognostic nomogram of LSCC patients involving ADH1B rs17033 was constructed. CONCLUSION: This study has demonstrated an independent association between ADH1B gene variants and the recurrence of LSCC. A nomogram model based on rs17033 of ADH1B, age, T, and N stages were successfully developed for the first time to predict the probability of recurrence in LSCC patients. LEVEL OF EVIDENCE: 3 Retrospective cohort study Laryngoscope, 132:2169-2176, 2022.

Oropharynx microbiota transitions in hypopharyngeal carcinoma treatment of induced chemotherapy followed by surgery.

AIMS: To analyze changes in oropharynx microbiota composition after receiving induced chemotherapy followed by surgery for hypopharyngeal squamous cell carcinoma (HPSCC) patients. METHODS: Clinical data and swab samples of 38 HPSCC patients (HPSCC group) and 30 patients with benign disease (control group, CG) were enrolled in the study. HPSCC group was stratified into two groups: induced chemotherapy group (IC) of 10 patients and non-induced chemotherapy group (nIC) of 28 patients. The microbiota from oropharyngeal membrane was analyzed through 16S rRNA sequencing. RESULTS: Alpha-diversity (Shannon and Ace indexes) and weighted UniFrac based beta-diversity severely decreased in the HPSCC group when compared with CG. In pre-operative comparisons, PCoA and NMDS analyses showed microbial structures in the IC group were more similar to CG than nIC. Both IC group and nIC group yielded significantly diverse post-operative communities in contrast to their pre-operative counterparts, evident by the decrease in genera Veillonella and Fusobacterium and increase in genera Streptococcus and Gemella. Given that post-operative oropharynx microbiota showed no difference between IC and nIC groups, the IC group showed less accumulation in anaerobic communities. The abundance of genera Fusobacterium, Parvimonas, Actinomyces were enhanced in the advanced stages (III/IV). CONCLUSIONS: Oropharynx microbiota in the HPSCC group presents dysbiosis with low diversity and abundance. Induced chemotherapy is beneficial in adjusting the oropharynx microbial environment leading to fewer amounts of anaerobe accumulation after operation. Higher amounts of Fusobacterium in advanced stages (III/IV) may influence the progression of HPSCC.

Glycolysis related gene expression signature in predicting prognosis of laryngeal squamous cell carcinoma.

Researches have suggested that aerobic glycolysis can reflect the development and progression of most carcinomas. We aimed to investigate whether glycolysis-related genes (GRGs) are associated with overall survival in laryngeal squamous cell carcinoma (LSCC). Here, we identified differentially expressed GRGs in TCGA dataset and microarray sample of GSE27020 from GEO database. A set of two glycolytic gene signatures, including DDIT4 and PLOD2 was screened through Cox and Lasso regression. The risk score was calculated using the gene expression of the two GRGs. The high-risk group presented a poor prognosis through Kaplan-Meier method. The ROC curve indicated good prediction performance in survival based on the validation of four cohorts. Univariate and multivariate Cox regression analyses suggested that two-gene signature could be an independent risk factor in LSCC. A total of 17 LSCC patients were enrolled to clarify the genetic expression through using reverse transcription-polymerase chain reaction (RT-PCR). A visualized nomogram was then constructed to predict 1-, 3-, and 5-year overall survival. Taken together, two novel glycolytic gene signatures were discovered and validated, providing a potential therapeutic and overall survival (OS)-prediction biomarker for LSCC.

Throat Microbial Community Structure and Functional Changes in Postsurgery Laryngeal Carcinoma Patients.

The microbial community structure in the throat and its shift after laryngectomy in laryngeal squamous cell carcinoma (LSCC) patients were investigated. Thirty swab samples taken prior to laryngectomy (SLC), 18 samples 1 week after laryngectomy (SLCA1w), and 30 samples 24 weeks after laryngectomy (SLCA24w) from 30 LSCC patients were examined. Microbial diversity was profiled through sequencing the V3-V4 variable region of the 16S rRNA gene. Quantitative real-time PCR (qPCR) was used to validate the 16S rRNA sequence data for the V3-V4 region. The community structure and function of throat microbiota were assessed by PICRUSt (phylogenetic investigation of communities by reconstruction of unobserved states) analysis. Both alpha and beta diversity results showed significant differences in the throat microbiota of LSCC patients before and after laryngectomy (P < 0.05). The drinking index of the SLC group was positively associated with the genus abundance of Prevotella (P < 0.05). The SLCA1w group had lower abundances of Fusobacterium, Leptotrichia, Lachnoanaerobaculum, and Veillonella than the SLC group (P < 0.05). The SLCA24w group had higher abundances of Streptococcus and Leptotrichia as well as lower abundances of Fusobacterium and Alloprevotella than the SLC group (P < 0.05). The throat microbiomes of the SLC group could be implicated in human cancer signaling pathways, as evidenced by PICRUSt analysis (P < 0.05). Our study clarifies alterations in throat microbial community structure and function in LSCC patients during the perioperative period and postoperative recovery period.IMPORTANCE Laryngeal squamous cell carcinoma greatly impacts patients' lives, and noninvasive means of prognostic assessment are valuable in determining the effectiveness of laryngectomy. We set out to study the microbial structure changes in the throat before and after laryngectomy and found the gene functions of several throat bacteria to be associated with human cancer signaling pathways. Our findings may offer insights into the disease management of patients with laryngeal squamous cell carcinoma. We hope to provide a means of using molecular mechanisms to improve the prognosis of laryngeal cancer treatment and to facilitate relevant research.

Prognostic values of preoperative platelet-to-lymphocyte ratio and platelet-related indices in advanced hypopharyngeal squamous cell carcinoma.

OBJECTIVES: To investigate the prognostic values of preoperative platelet-to-lymphocyte ratio (PLR) and platelet-related indices in advanced hypopharyngeal squamous cell carcinoma (HPSCC). METHODS: The data of 247 eligible advanced HPSCC patients were reviewed retrospectively. Pretreatment haematological parameters were categorised into two groups based on the result of X-tile, and several variates were assessed using chi-square test, Kaplan-Meier method, Cox univariate and multivariate analysis. RESULTS: The optimal cut-off points of 171.4 for PLR, 260 × 109 /L for platelet, 10.4 fL for mean platelet volume (MPV) and 16.5% for platelet distribution width were defined. The haematological parameters PLR and MPV, postoperative metastasis and internal jugular vein invasion were statistically significant in OS and DFS analyses (P < .05). The high PLR (>171.4) or high MPV (>10.4 fL) was significantly associated with worse OS and DFS (P < .05). CONCLUSIONS: The preoperative levels of PLR and MPV could be considered as independent prognostic predictors in patients with advanced HPSCC.

Pretreatment Level of Red Cell Distribution Width as a Prognostic Indicator for Survival in a Large Cohort Study of Male Laryngeal Squamous Carcinoma.

Objective: High levels of red cell distribution width (RDW) may be associated with adverse outcomes in patients with cancer. The purpose of the present study was to investigate the prognostic impact of pretreatment RDW levels on overall survival (OS), cancer-specific survival (CSS), and disease-free survival (DFS) in a large cohort of male laryngeal squamous cell cancer (LSCC) patients. Methods: A total of 809 LSCC patients who were treated between 2007 and 2011 at the Eye & ENT Hospital of Fudan University were enrolled and evaluated retrospectively. OS, CSS, and DFS were analyzed using the Kaplan-Meier method. To evaluate the prognostic significance of RDW levels, univariate, and multivariate Cox analyses were applied. Results: Higher pretreatment RDW levels were significantly associated with high death events, red blood cell count, hemoglobin, radiotherapy, operation therapy, and advanced tumor stage (p < 0.05). From the univariate analysis, we observed that the higher (13.2-13.5%) and the highest (>13.5%) quartiles of RDW level were consistent factors for poor OS, CSS, and DFS in LSCC patients. In the multivariate analysis, after adjusting for confounding factors, the higher and highest quartiles of RDW levels were identified as independent prognostic factors in male LSCC patients. Conclusion: Higher pretreatment RDW levels were demonstrated to be associated with poor clinical outcome in male LSCC patients and might be novel markers for patient stratification in LSCC management.