Head And Neck Squamous Cell Carcinoma Tissues Research Articles

MicroRNA-2355-5p Promotes the Proliferation of Head and Neck Squamous Cell Carcinoma via Suppressing NISCH Expression

Background. MicroRNAs (miRNAs) have emerged as crucial regulators in various cancers. However, the potential role of miR-2355-5p in head and neck squamous cell carcinoma (HNSC) remains unclear. Methods. Bioinformatics methods were implemented to find the candidate target gene of miR-2355-5p. Quantitative real-time PCR was performed to detect RNA expression levels of miR-2355-5p and NISCH, while western blot was carried out for the detection of protein levels of NISCH and cell cycle-related biomarkers. CCK-8, EdU staining, and flow cytometry were employed to measure cell proliferation and cell cycle progression. Dual-luciferase assay and RNA pulldown were conducted to verify the binding relationship between miR-2355-5p and NISCH. Results. The expression levels of miR-2355-5p and NISCH were, respectively, higher and lower in HNSC tissues than those in normal adjacent tissues. The transfection of the miR-2355-5p inhibitor suppressed cell proliferation by arresting the cells at the G1/S transition. The results of luciferase activity and RNA pulldown assays indicated that miR-2355-5p directly targeted the NISCH 3′-untranslated region. Furthermore, the effects of miR-2355-5p inhibition on cell proliferation were reversed after treatment with siRNA against NISCH. Conclusion. In summary, our findings indicate that miR-2355-5p promotes cell cycle progression in HNSC by targeting NISCH. Hence, targeting miR-2355-5p could be a promising therapeutic strategy for the treatment of HNSC

SPDEF suppresses head and neck squamous cell carcinoma progression by transcriptionally activating NR4A1

SAM pointed domain containing E26 transformation-specific transcription factor (SPDEF) plays dual roles in the initiation and development of human malignancies. However, the biological role of SPDEF in head and neck squamous cell carcinoma (HNSCC) remains unclear. In this study, the expression level of SPDEF and its correlation with the clinical parameters of patients with HNSCC were determined using TCGA-HNSC, GSE65858, and our own clinical cohorts. CCK8, colony formation, cell cycle analysis, and a xenograft tumor growth model were used to determine the molecular functions of SPDEF in HNSCC. ChIP-qPCR, dual luciferase reporter assay, and rescue experiments were conducted to explore the potential molecular mechanism of SPDEF in HNSCC. Compared with normal epithelial tissues, SPDEF was significantly downregulated in HNSCC tissues. Patients with HNSCC with low SPDEF mRNA levels exhibited poor clinical outcomes. Restoring SPDEF inhibited HNSCC cell viability and colony formation and induced G0/G1 cell cycle arrest, while silencing SPDEF promoted cell proliferation in vitro. The xenograft tumor growth model showed that tumors with SPDEF overexpression had slower growth rates, smaller volumes, and lower weights. SPDEF could directly bind to the promoter region of NR4A1 and promoted its transcription, inducing the suppression of AKT, MAPK, and NF-κB signaling pathways. Moreover, silencing NR4A1 blocked the suppressive effect of SPDEF in HNSCC cells. Here, we demonstrate that SPDEF acts as a tumor suppressor by transcriptionally activating NR4A1 in HNSCC. Our findings provide novel insights into the molecular mechanism of SPDEF in tumorigenesis and a novel potential therapeutic target for HNSCC.

Identification of key genes for HNSCC from public databases using bioinformatics analysis

BackgroundThe cause and underlying molecular mechanisms of head and neck squamous cell carcinoma (HNSCC) are unclear. Our study aims to identify the key genes associated with HNSCC and reveal potential biomarkers.MethodsIn this study, the expression profile dataset GSE83519 of the Gene Expression Omnibus database and the RNA sequencing dataset of HNSCC of The Cancer Genome Atlas were included for analysis. Sixteen differentially expressed genes were screened from these two datasets using R software. Gene Expression Profiling Interactive Analysis 2 (GEPIA2) was then adopted for survival analysis, and finally, three key genes related to the overall survival of HNSCC patients were identified. Furthermore, we verified these three genes using the Oncomine database and from real-time PCR and immunohistochemistry results from HNSCC tissues.ResultsThe expression data of 44 samples from GSE83519 and 545 samples from TCGA-HNSC were collected. Using bioinformatics, the two databases were integrated, and 16 DEGs were screened out. Gene Ontology (GO) enrichment analysis showed that the biological functions of DEGs focused primarily on the apical plasma membrane and regulation of anoikis. Kyoto Encyclopedia of Genes and Genomes (KEGG) signalling pathway analysis showed that these DEGs were mainly involved in drug metabolism-cytochrome P450 and serotonergic synapses. Survival analysis identified three key genes, CEACAM5, CEACAM6 and CLCA4, that were closely related to HNSCC prognosis. The Oncomine database, qRT–PCR and IHC verified that all 3 key genes were downregulated in most HNSCC tissues compared to adjacent normal tissues.ConclusionsThis study indicates that integrated bioinformatics analyses play an important role in screening for differentially expressed genes and pathways in HNSCC, helping us better understand the biomarkers and molecular mechanism of HNSCC.

Upregulation of HOX genes promotes cell migration and proliferation in head and neck squamous cell carcinoma.

Expression dysregulation of HOX homeobox genes has been observed in several cancers, including head and neck squamous cell carcinoma (HNSC). Although characterization of HOX gene roles in HNSC development has been reported, there is still a need to better understand their real contribution to tumorigenesis. The present study aimed to evaluate the contribution of the protein-coding HOX genes (HOXA10, HOXC9, HOXC10, and HOXC13) in cellular processes related to carcinogenesis and progression of the HNSC. Expression of HOX genes was analyzed in HNSC RNA-Seq data from The Cancer Genome Atlas (TCGA) and by RT-qPCR in different tumor cell lines. siRNA-mediated knockdown of HOXA10, HOXC9, HOXC10 or HOXC13 was performed in HNSC cell lines, and predicted transcriptional targets HOX genes was analyzed by bioinformatic. Thirty-one out of the 39 mammalian HOX genes were found upregulated in HNSC tissues and cell lines. The HOXC9, HOXC10 or HOXC13 knockdown attenuated cell migration, and lead to downregulation of epithelial-mesenchymal transition (EMT) markers, which were predicted as transcriptional targets of these three HOX genes. Diminished colony formation and cell cycle arrest after HOXC10 or HOXC13 knockdown were also observed, corroborating the fact that there was an enrichment for genes in proliferation/cell cycle pathways. In summary, we revealed roles for HOXC9, HOXC10, and HOXC13 in cell migration and proliferation/cell cycle progression in HNSC cells and suggested that those HOX members contribute to HNSC development possibly by regulating tumor growth and metastasis.

MiR-17-5p drives G2/M-phase accumulation by directly targeting CCNG2 and is related to recurrence of head and neck squamous cell carcinoma

BackgroundThe human miR-17-92 polycistron is the first reported and most well-studied onco-miRNA with a cluster of seven miRNAs. miR-17-5p, a member of the miR-17-92 family, plays an important role in tumor cell proliferation, apoptosis, migration and invasion. However, few studies have shown the role of miR-17-5p in the cell cycle of head and neck squamous cell carcinoma (HNSCC).MethodsRT-qPCR was used to detect miR-17-5p expression levels in 64 HNSCC tissues and 5 cell lines. The relationship between the expression of miR-17-5p in the tissues and the clinical characteristics of the patients was analyzed. HNSCC cells were transfected with an miR-17-5p mimic or inhibitor to evaluate cell cycle distribution by flow cytometry. Cell cycle distribution of cells transfected with target gene was evaluated using flow cytometry. Dual-luciferase reporter assay was used to detect the regulatory effect of miR-17-5p on target gene expression.ResultsIn the present study, we found that miR-17-5p expression in HNSCC tissues and cell lines was remarkably increased, and miR-17-5p is related to recurrence in HNSCC patients. Silencing miR-17-5p blocked HNSCC cells in G2/M phase, whereas its overexpression propelled cell cycle progression. More importantly, we verified that miR-17-5p negatively regulated CCNG2 mRNA and protein expression by directly targeting its 3’UTR.ConclusionThese findings suggest that miR-17-5p might act as a tumor promoter and prognostic factor for recurrence in HNSCC patients.

Melatonin modulates metabolic remodeling in HNSCC by suppressing MTHFD1L-formate axis.

Metabolic remodeling is now widely recognized as a hallmark of cancer, yet its role in head and neck squamous cell carcinoma (HNSCC) remains largely unknown. In this study, metabolomic analysis of melatonin-treated HNSCC cell lines revealed that exogenous melatonin inhibited many important metabolic pathways including folate cycle in HNSCC cells. Methylenetetrahydrofolate dehydrogenase 1 like (MTHFD1L), a metabolic enzyme of the folate cycle regulating the production of formate, was identified as a downstream target of melatonin. MTHFD1L was found to be markedly upregulated in HNSCC, and MTHFD1L overexpression was significantly associated with unfavorable clinical outcome of HNSCC patients. In addition, MTHFD1L promoted HNSCC progression in vitro and in vivo and reversed the oncostatic effects of exogenous melatonin. More importantly, the malignant phenotypes suppressed by knockdown of MTHFD1L or exogenous melatonin could be partially rescued by formate. Furthermore, we found that melatonin inhibited the expression of MTHFD1L in HNSCC cells through the downregulation of cyclic AMP-responsive element-binding protein 1 (CREB1) phosphorylation. Lastly, this novel regulatory axis of melatonin-p-CREB1-MTHFD1L-formate was also verified in HNSCC tissues. Collectively, our findings have demonstrated that MTHFD1L-formate axis promotes HNSCC progression and melatonin inhibits HNSCC progression through CREB1-mediated downregulation of MTHFD1L and formate. These findings have revealed new metabolic mechanisms in HNSCC and may provide novel insights on the therapeutic intervention of HNSCC.

PD-L2 glycosylation promotes immune evasion and predicts anti-EGFR efficacy

BackgroundCombination therapy has been explored for advanced head and neck squamous cell carcinoma (HNSCC) owing to the limited efficacy of anti-epidermal growth factor receptor (EGFR) therapy. Increased expression and glycosylation...

The expression profile and clinical application value of hsa_circ_0016148 in head and neck squamous cell carcinoma.

BackgroundDysregulated circular RNAs (circRNAs) are involved in human cancers and may be used as biomarkers with the potential of clinical application. However, little is known regarding the mechanism of circRNAs and their clinical application value in head and neck squamous cell carcinoma (HNSCC).MethodsIn the current study, we established the profile of circRNAs in HNSCC using microarray and then measured the expression of hsa_circ_0016148 in 137 paired HNSCC tissues by qRT‐PCR technique, analyzed the relationship between hsa_circ_0016148 and clinicopathological data, and investigated its diagnostic and prognostic value. The hsa_circ_0016148‐miRNA‐mRNA interaction network was predicted and constructed by Cytoscape.ResultsOur study showed a circRNA expression profile and confirmed downregulated hsa_circ_0016148 in HNSCC tissues (p = 2.64E‐35). The hsa_circ_0016148 expression is remarkably correlated with lymph node metastasis (p = 0.001) and clinical stage (p = 0.029). Then, the area under the receiver characteristic curve (AUC) was 0.912 with 92% of sensitivity and 86.9% specificity, respectively. Besides, our study demonstrated that lower‐expressed hsa_circ_0016148 could independently predict poorer overall survival of HNSCC patients (hazard ratio [HR] = 0.456; 95% confidence interval [CI] = 0.265–0.784; p = 0.005). The hsa_circ_0016148‐miRNA‐mRNA interaction network was constructed, which included a total of nine targeted miRNAs.ConclusionTaken together, our results revealed that hsa_circ_0016148 might play a critical role in HNSCC tumorigenesis and may serve as an indicator with the potential of diagnosis and prognosis for HNSCC.

Identification of a Novel Signature Predicting Overall Survival in Head and Neck Squamous Cell Carcinoma.

Background: Head and neck squamous cell carcinoma (HNSCC) is a highly heterogeneous tumor with a high incidence and poor prognosis. Therefore, effective predictive models are needed to evaluate patient outcomes and optimize treatment.Methods: Robust Rank Aggregation (RRA) method was used to identify highly robust differentially-expressed genes (DEGs) between HNSCC and normal tissue in 9 GEO and TCGA datasets. Univariate Cox regression analysis and Lasso Cox regression analysis were performed to identify DEGs related to the Overall survival (OS) and to construct a prognostic gene signature (HNSCCSig). External validation was performed using GSE65858 dataset. Moreover, comprehensive bioinformatics analyses were used to identify the association between HNSCCSig and tumor immune environment.Results: A total of 257 reliable DEGs were identified by differentially analysis result of TCGA and GSE65858 datasets. The HNSCCSig including 7 mRNAs (SLURP1, SCARA5, CLDN10, MYH11, CXCL13, HLF, and ITGA3) were developed and validated to identify high-risk group who had a worse OS than low-risk group in TCGA and GSE65858 datasets. Cox regression analysis showed that the HNSCCSig could independently predict OS in both the TCGA and the GSE65858 datasets. Further research demonstrated that the infiltration bundance of CD8 + T cells, B cells, neutrophils, and NK cells were significantly lower in the high-risk group. A nomogram was also constructed by combining the HNSCCSig and clinical characters.Conclusion: We established and validated the HNSCCSig consisting of SLURP1, SCARA5, CLDN10, MYH11, CXCL13, HLF, and ITGA3. A nomogram combining HNSCCSig and some clinical parameters was constructed to identify high-risk HNSCC-patients with poor prognosis.

Impact of Oncogenic Targets by Tumor-Suppressive miR-139-5p and miR-139-3p Regulation in Head and Neck Squamous Cell Carcinoma.

We newly generated an RNA-sequencing-based microRNA (miRNA) expression signature of head and neck squamous cell carcinoma (HNSCC). Analysis of the signature revealed that both strands of some miRNAs, including miR-139-5p (the guide strand) and miR-139-3p (the passenger strand) of miR-139, were downregulated in HNSCC tissues. Analysis of The Cancer Genome Atlas confirmed the low expression levels of miR-139 in HNSCC. Ectopic expression of these miRNAs attenuated the characteristics of cancer cell aggressiveness (e.g., cell proliferation, migration, and invasion). Our in silico analyses revealed a total of 28 putative targets regulated by pre-miR-139 (miR-139-5p and miR-139-3p) in HNSCC cells. Of these, the GNA12 (guanine nucleotide-binding protein subunit alpha-12) and OLR1 (oxidized low-density lipoprotein receptor 1) expression levels were identified as independent factors that predicted patient survival according to multivariate Cox regression analyses (p = 0.0018 and p = 0.0104, respectively). Direct regulation of GNA12 and OLR1 by miR-139-3p in HNSCC cells was confirmed through luciferase reporter assays. Moreover, overexpression of GNA12 and OLR1 was detected in clinical specimens of HNSCC through immunostaining. The involvement of miR-139-3p (the passenger strand) in the oncogenesis of HNSCC is a new concept in cancer biology. Our miRNA-based strategy will increase knowledge on the molecular pathogenesis of HNSCC.

A novel intronic circular RNA, circGNG7, inhibits head and neck squamous cell carcinoma progression by blocking the phosphorylation of heat shock protein 27 at Ser78 and Ser82.

BackgroundThere is increasing evidence that circular RNAs (circRNAs) play a significant role in pathological processes including tumorigenesis. In contrast to exonic circRNAs, which are the most frequently reported circRNAs in cancer so far, the studies of intronic circRNAs have been greatly lagged behind. Here, we aimed to investigate the regulatory role of intronic circRNAs in head and neck squamous cell carcinoma (HNSCC).MethodsWe conducted whole‐transcriptome sequencing with four pairs of primary tumor tissues and adjacent normal tissues from HNSCC patients. Then, we characterized circGNG7 expression in HNSCC tissues and cell lines and explored its association with the prognosis of HNSCC patients. We also identified interactions between circGNG7 and functional proteins, which alter downstream signaling that regulate HNSCC progression.ResultsIn this study, we identified a new intronic circRNA, circGNG7, and validated its functional roles in HNSCC progression. CircGNG7 was predominately localized to the cytoplasm, and its expression was downregulated in both HNSCC tissues andCAL27, CAL33, SCC4, SCC9, HN6, and HN30 cells. Low expression of circGNG7 was significantly correlated with poor prognosis in HNSCC patients. Consistent with this finding, overexpression of circGNG7 strongly inhibited tumor cell proliferation, colony formation, in vitro migration, and in vivo tumor growth. Mechanistically, the expression of circGNG7 in HNSCC cells was regulated by the transcription factor SMAD family member 4 (SMAD4). Importantly, we discovered that circGNG7 could bind to serine residues 78 and 82 of the functional heat shock protein 27 (HSP27), occupying its phosphorylation sites and hindering its phosphorylation, which reduced HSP27‐JNK/P38 mitogen‐activated protein kinase (MAPK) oncogenic signaling. Downregulation of circGNG7 expression in HNSCC increased HSP27‐JNK/P38 MAPK signaling and promoted tumor progression.ConclusionsOur results revealed that a new intronic circRNA, circGNG7, functions as a strong tumor suppressor and that circGNG7/HSP27‐JNK/P38 MAPK signaling is a novel mechanism by which HNSCC progression can be controlled.

The role of SPP1 as a prognostic biomarker and therapeutic target in head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is one of the most common malignancies and has a low 5-year survival rate. Mounting evidence suggests that oral potentially malignant disorders, such as oral leukoplakia (OLK), may progress to HNSCC. Given that OLK and HNSCC are often insidious and asymptomatic, the identification of markers of OLK malignant transformation and therapeutic targets in HNSCC is critical. Using various online tools and publicly available gene expression datasets, the secreted phosphoprotein 1 gene (SPP1) was identified as a significant differentially expressed gene among OLK, HNSCC, and non-cancerous tissues. SPP1 mRNA levels were elevated in HNSCC tissues and were associated with cancer stage, tumor grade, and human papillomavirus infection status. High SPP1 mRNA levels were correlated with poor overall survival of HNSCC patients. In contrast, SPP1 mutations were not significantly associated with overall survival, although their frequency in HNSCC was very low (0.6%). Furthermore, SPP1 expression levels in HNSCC were positively correlated with the infiltration of CD4+ cells, macrophages, neutrophils, and dendritic cells. The study results suggest that SPP1 may represent a diagnostic and prognostic biomarker, as well as a potential therapeutic target in HNSCC.

Prognostic role of DFNA5 in head and neck squamous cell carcinoma revealed by systematic expression analysis

BackgroundThe gasdermin E gene (GSDME, also known as DFNA5) is mutated in familial aging-related hearing loss. Recent studies have also revealed that the expression of DFNA5 is suppressed in many cancer types; however, little is known about the function of DFNA5 in head and neck squamous cell carcinoma (HNSCC). Accordingly, the aim of the present study was to evaluate the expression of DFNA5 and explore its prognostic value in HNSCC.ResultWe used a set of bioinformatics tools, including Oncomine, TIMER, TISIDB, cBioPortal, and GEPIA, to analyze the expression of DFNA5 in patients with HNSCC from public databases. Kaplan-Meier plotter was used to evaluate the potential prognostic significance of DFNA5. DFNA5 mRNA levels were significantly higher in HNSCC tissues than in normal tissues, and high DFNA5 expression was correlated with worse survival. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses showed that DFNA5 expression has a strong positive correlation with cell adhesion and the integrin signaling pathway, whereas its expression was negatively correlated with the levels of infiltrating B cells (cor = − 0.223, P = 8.57e-07) and CD8 T cells (cor = − 0.223, P = 2.99e-07).ConclusionThis study demonstrates that DFNA5 expression has prognostic value for HNSCC patients. Moreover, these results suggest that regulation of lymphocyte infiltration is the mechanism underlying the function of DFNA5 in HNSCC.

A 14-Marker Multiplexed Imaging Panel for Prognostic Biomarkers and Tumor Heterogeneity in Head and Neck Squamous Cell Carcinoma

Recent advances made in treatment for head and neck squamous cell carcinoma (HNSCC) highlight the need for new prediction tools to guide therapeutic strategies. In this study, we aimed to develop a HNSCC-targeting multiplex immunohistochemical (IHC) panel that can evaluate prognostic factors and the intratumor heterogeneity of HNSCC. To identify IHC-based tissue biomarkers that constitute new multiplex IHC panel, a systematic review and meta-analysis were performed to analyze reported IHC biomarkers in laryngeal and pharyngeal SCC in the period of 2008–2018. The Cancer Genome Atlas (TCGA) and Reactome pathway databases were used to validate the prognostic and functional significance of the identified biomarkers. A 14-marker chromogenic multiplex IHC panel including identified biomarkers was used to analyze untreated HNSCC tissue. Forty-five high-quality studies and thirty-one candidate tissue biomarkers were identified (N = 7062). Prognostic validation in TCGA laryngeal and pharyngeal SCC cohort (N = 205) showed that β-catenin, DKK1, PINCH1, ADAM10, and TIMP1 were significantly associated with poor prognosis, which were related to functional categories such as immune system, cellular response, cell cycle, and developmental systems. Selected biomarkers were assembled to build a 14-marker panel, evaluating heterogeneity and polarized expression of tumor biomarkers in the tissue structures, which was particularly related to activation of Wnt/β-catenin pathway. Integrated IHC analysis based on a systemic review and meta-analysis provides an in situ proteomics tool to assess the aggressiveness and intratumor heterogeneity of HNSCC.

FOXM1 Promotes Head and Neck Squamous Cell Carcinoma via Activation of the Linc-ROR/LMO4/AKT/PI3K Axis.

Background/AimPrevious literature has implicated the sustained expression of FOXM1 in numerous human cancers, including head and neck squamous cell carcinoma (HNSCC). The current study aimed to elucidate the function and regulatory mechanism of FOXM1 in HNSCC.MethodsWestern blot and RT-qPCR methods were performed to evaluate the expression of Linc-ROR, FOXM1, and LMO4 in HNSCC tissue samples and cells. The binding between FOXM1 and Linc-ROR was analyzed using a ChIP assay. Various cellular processes including proliferation and invasion abilities were assessed following alteration of FOXM1, Linc-ROR and LMO4 expression in HNSCC cells. Xenograft mouse models were established to validate the in vitro findings.ResultsLinc-ROR and FOXM1 were highly expressed in HNSCC tissues and cells. FOXM1 operated as a potential transcription factor to bind to the promoter region of Linc-ROR. Linc-ROR and FOXM1 exhibited high expression levels in both the clinical tissue samples as well as the HNSCC cells, which could facilitate the proliferation and invasion of HNSCC cells. Linc-ROR upregulated the expression of LMO4 and promoted activation of the AKT/PI3K signaling pathway, thus stimulating the proliferation and invasion of HNSCC cells. Silencing of Linc-ROR brought about a contrasting effect relative to that seen when FOXM1 was overexpressed in HNSCC in vivo.ConclusionsOverall, FOXM1 promoted the expression of Linc-ROR and induced the activation of the LMO4-dependent AKT/PI3K signaling pathway, thus facilitating the occurrence and development of HNSCC.

Alternatively spliced ANLN isoforms synergistically contribute to the progression of head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is a common cancer with high mortality. Anilin actin-binding protein (ANLN) has been reported to be associated with carcinogenesis in multiple tumors. However, the expression pattern and functional effects of ANLN in HNSCC remain to be unclear. Clinical data and online databases were used to analyze the expression of ANLN and its relationship with HNSCC patient survival. Expression of two major splice variants of ANLN was assessed in HNSCC tissues and cell lines. The functional effects and related mechanisms of ANLN isoforms were investigated in HNSCC in vitro and in vivo. Our study showed that patients with high expression of ANLN had a poor prognosis. The two primary isoforms of ANLN transcripts ANLN-201 and ANLN-210 were highly expressed in HNSCC tissues and cell lines. Knockout of ANLN restrained cell proliferation, migration, and invasion of SCC-9 cells. Mechanically, ANLN-201 could interact with c-Myc to keep its protein stability, thereby playing a oncogenic role in HNSCC. ANLN-210 could be transferred to macrophages via exosomes by binding to RNA-binding protein hnRNPC. Exosomal ANLN-210 promoted macrophage polarization via PTEN/PI3K/Akt signaling pathway, thus stimulating tumor growth of HNSCC. ANLN was an independent prognostic factor in patients with HNSCC. Alternatively spliced ANLN isoforms collaboratively promote HNSCC tumorigenesis in vitro and in vivo, which might provide the in-depth role and mechanism of ANLN in HNSCC development.

An aberrant DNA methylation signature for predicting the prognosis of head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a common malignancy worldwide with a poor prognosis. DNA methylation is an epigenetic modification that plays a critical role in the etiology and pathogenesis of HNSCC. The current study aimed to develop a predictive methylation signature based on bioinformatics analysis to improve the prognosis and optimize therapeutic outcome in HNSCC. Clinical information and methylation sequencing data of patients with HNSCC were downloaded from The Cancer Genome Atlas database. The R package was used to identify differentially methylated genes (DMGs) between HNSCC and adjacent normal tissues. We identified 22 DMGs associated with 246 differentially methylated sites. Patients with HNSCC were classified into training and test groups. Cox regression analysis was used to build a risk score formula based on the five methylation sites (cg26428455, cg13754259, cg17421709, cg19229344, and cg11668749) in the training group. The Kaplan–Meier survival curves showed that the overall survival (OS) rates were significantly different between the high‐ and low‐risk groups sorted by the signature in the training group (median: 1.38 vs. 1.57 years, log‐rank test, p < 0.001). The predictive power was then validated in the test group (median: 1.34 vs. 1.75 years, log‐rank test, p < 0.001). The area under the receiver operating characteristic curve (area under the curve) based on the signature for predicting the 5‐year survival rates, was 0.7 in the training and 0.73 in test groups, respectively. The results of multivariate Cox regression analysis showed that the riskscore (RS) signature based on the five methylation sites was an independent prognostic tool for OS prediction in patients. In addition, a predictive nomogram model that incorporated the RS signature and patient clinical information was developed. The innovative methylation signature‐based model developed in our study represents a robust prognostic tool for guiding clinical therapy and predicting the OS in patients with HNSCC.

Lactate secreted by PKM2 upregulation promotes Galectin-9-mediated immunosuppression via inhibiting NF-\u03baB pathway in HNSCC

Pyruvate kinase M2 as a key rate-limiting enzyme in glycolysis, it plays a critical role in metabolic reprogramming and carcinogenesis. However, whether PKM2 can promote immunosuppressive microenvironment formation remains unknown in head and neck squamous cell carcinoma (HNSCC). PKM2 expression was detected using immunohistochemical staining. The biological functions of PKM2 were investigated in vitro and in vivo. Lactate production and the expression of Galectin-9, a critical immunosuppression molecule, were detected after PKM2 knockdown and overexpression in HNSCC cells. The mechanism of lactate regulating Galectin-9 expression through NF-κB signaling was explored in vitro. Overexpression of PKM2 correlates with poor prognosis in HNSCC patients. Silencing PKM2 markedly inhibits proliferation and metastasis capacity in vivo and in vitro, and vice versa. The glycolysis and glycolytic capacity are significantly decreased after PKM2 silencing. Lactate secretion induced by PKM2 significantly promotes migration and invasion capacity. Furthermore, a positive correlation between PKM2 and Galectin-9 expression is observed in HNSCC tissues. The induction of Galectin-9 expression by PKM2 can be affected by a lactate transporter inhibitor. Mechanically, lactate impeded the suppressive transcriptional complex formation of NF-κB and histone deacetylase 3 (HDAC3), which released the transcription of Galectin-9 mediated by NF-κB signaling. Our findings demonstrate that lactate produced by PKM2 upregulation promotes tumor progression and Galectin-9-mediated immunosuppression via NF-κB signaling inhibition in HNSCC, which bridges metabolism and immunosuppression. The novel PKM2-lactate-Galectin-9 axis might be a potential therapeutic target in HNSCC.

Identification and Confirmation of the miR-30 Family as a Potential Central Player in Tobacco-Related Head and Neck Squamous Cell Carcinoma.

Constituents of tobacco that can cause DNA adduct formation and oxidative stress are implicated in the development of head and neck squamous cell carcinoma (HNSCC). However, there are few studies on the mechanism(s) that underlie tobacco-associated HNSCC. Here, we used a model in which tumors were induced in rats using 4-nitroquinoline 1-oxide (4NQO), which mimicked tobacco-related HNSCC, and analyzed the expression profiles of microRNAs and mRNAs. Our results indicated that 57 miRNAs and 474 mRNA/EST transcripts exhibited differential expression profiles between tumor and normal tongue tissues. In tumor tissue, the expression levels of rno-miR-30 family members (rno-miR-30a, rno-miR-30a-3p, rno-miR-30b-5p, rno-miR-30c, rno-miR-30d, rno-miR-30e and rno-miR-30e-3p) were only 8% to 37% of those in the control group. The GO terms enrichment analysis of the differentially expressed miRNAs indicated that oxidation reduction was the most enriched process. Low expression of miR-30 family members in human HNSCC cell lines and tissues was validated by qPCR. The results revealed that the expression of miR-30b-5p and miR-30e-5p was significantly decreased in the TCGA HNSCC dataset and validation datasets, and this decrease in expression further distinguishes HNSCC associated with tobacco use from other subtypes of HNSCC. CCK8, colony formation, transwell migration and HNSCC xenograft tumor assays indicated that miR-30b-5p or miR-30e-5p inhibited proliferation, migration and invasion in vitro, and miR-30b-5p suppressed tumor growth in vivo. Moreover, we uncovered that KRAS might be the potential target gene of miR-30e-5p or miR-30b-5p. Thus, our data clearly showed that decreased expression of miR-30e-5p or miR-30b-5p may play a crucial role in cancer development, especially that of tobacco-induced HNSCC, and may be a novel candidate biomarker and target for this HNSCC subtype.

LncRNA lnc-POP1-1 upregulated by VN1R5 promotes cisplatin resistance in head and neck squamous cell carcinoma through interaction with MCM5

Cisplatin resistance is a major therapeutic challenge in advanced head and neck squamous cell carcinoma (HNSCC). Here, we aimed to investigate the key signaling pathway for cisplatin resistance in HNSCC cells. Vomeronasal type-1 receptor 5 (VN1R5) was identified as a cisplatin resistance-related protein and was highly expressed in cisplatin-resistant HNSCC cells and tissues. The long noncoding RNA (lncRNA) lnc-POP1-1 was confirmed to be a downstream target induced by VN1R5. VN1R5 transcriptionally regulated lnc-POP1-1 expression by activating the specificity protein 1 (Sp1) transcription factor via the cyclic AMP (cAMP)/protein kinase A (PKA) pathway. VN1R5 promoted cisplatin resistance in HNSCC cells in a lnc-POP1-1-dependent manner. Mechanistically, lnc-POP1-1 bound to the minichromosome maintenance deficient 5 (MCM5) protein directly and decelerated MCM5 degradation by inhibiting ubiquitination of the MCM5 protein, which facilitated the repair of DNA damage caused bycisplatin. In summary, we identified the cisplatin resistance-related protein VN1R5 and its downstream target lnc-POP1-1. Upon upregulation by VN1R5, lnc-POP1-1 promotes DNA repair in HNSCC cells through interaction with MCM5 and deceleration of its degradation.