Head And Neck Squamous Cell Carcinoma Tumor Tissues Research Articles

CELSR3 is a prognostic marker in HNSCC and correlates with immune cell infiltration in the tumor microenvironment

PurposeTo look at the diagnostic value of the CELSR receptor 3 (CELSR3) gene in head and neck squamous cell carcinoma (HNSCC) and its effect on tumor immune invasion, which is important for enhancing HNSCC treatment.MethodsSeveral bioinformatics tools were employed to investigate CELSR3’s putative oncogenic pathway in HNSCC, and datasets from The Tumor Genome Atlas (TCGA), Tumor Immune Estimation Resource (TIMER), Gene Expression Profile Interaction Analysis (GEPIA) and LinkedOmics were extracted and evaluated. CELSR3 has been linked to tumor immune cell infiltration, immunological checkpoints, and immune-related genes. CELSR3’s putative roles were investigated using Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and pathway enrichment analysis. The expression level of CELSR3 in HNSCC tissues and cells was detected by RT-qPCR. The effects of CELSR3 on proliferation of HNSCC cells were detected by CCK-8 assay.ResultsCELSR3 was shown to be expressed differently in different types of cancer and normal tissues. CELSR3 gene expression was linked to pN-stage and pM-stage. Patients with high CELSR3 expression also have a well prognosis. CELSR3 expression was found to be an independent predictive factor for HNSCC in both univariate and multivariate Cox regression analyses. We discovered the functional network of CELSR3 in HNSCC using GO and KEGG analysis. CELSR3 expression levels were found to be favorably associated with immune cell infiltration levels. Furthermore, CELSR3 expression levels were significantly correlated with the expression levels of many immune molecules, such as MHC genes, immune activation genes, chemokine receptors, and chemokines. CELSR3 is highly expressed in HNSCC tissues and cells. CELSR3 overexpression significantly inhibited the proliferation of HNSCC cells. CELSR3 expression may affect the immune microenvironment and, as a result, the prognosis of HNSCC.ConclusionCELSR3 expression is elevated in HNSCC tumor tissues, and high CELSR3 expression is associated with well prognosis, which inhibited the proliferation of NHSCC cells. CELSR3 has the potential to influence tumor formation by controlling tumor-infiltrating cells in the tumor microenvironment (TME). As a result, CELSR3 may have diagnostic significance in HNSCC.

Nicotine downregulates miR-375–3p via neurotrophic tyrosine receptor kinase 2 to enhance the malignant behaviors of laryngopharyngeal squamous epithelial cells

Nicotine exposure from smoking constitutes a significant global public health concern. Furthermore, smoking represents a pivotal risk factor for head and neck squamous cell carcinoma (HNSCC). However, the influence of nicotine on HNSCC remains relatively underexplored. Our aim was to unravel the molecular mechanisms that underlie the effect of nicotine on the metastatic cascade of HNSCC. In this study, we discovered a significant association between smoking and HNSCC metastasis and prognosis. Nicotine significantly enhanced HNSCC cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro. Analysis of TCGA-HNSCC and FDEENT-HNSCC cohorts revealed reduced miR-375–3p levels in HNSCC tumor tissues, particularly among current smokers. Additionally, miR-375–3p level was strongly correlated with both lymph node metastasis and tumor stage. By downregulating miR-375–3p, nicotine promotes HNSCC cell metastasis in vitro and hematogenous metastatic capacity in vivo. Utilizing transcriptomic sequencing, molecular docking, dual-luciferase reporter assay, and fluorescence in situ hybridization (FISH), we demonstrated that miR-375–3p specifically binds to 3′ untranslated region (3’UTR) of NTRK2 mRNA. Thus, this study uncovers a novel nicotine-induced mechanism involving miR-375–3p-mediated NTRK2 targeting, which promotes HNSCC metastasis. These findings have implications for improving the prognosis of patients with HNSCC, especially in smokers.

USP39 Promotes the Viability and Migration of Head and Neck Squamous Cell Carcinoma Cell by Regulating STAT1.

Objective: Ubiquitin-specific peptidase 39 (USP39) plays a carcinogenic role in many cancers, but little research has been conducted examining whether it is involved in head and neck squamous cell carcinoma (HNSCC). Therefore, this study explored the functional role of USP39 in HNSCC. Method: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify differentially expressed proteins (DEPs) between the HNSCC tumor and adjacent healthy tissues. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were used to assess the functional enrichment of DEPs. Immunohistochemistry was used to detect protein expression. The viability and migration of two HNSCC cell lines, namely CAL27 and SCC25, were detected using the cell counting kit-8 assay and a wound healing assay, respectively. Quantitative real-time PCR was used to detect the expression level of signal transducer and activator of transcription 1 (STAT1) mRNA. Results: LC-MS/MS results identified 590 DEPs between HNSCC and adjacent tissues collected from 4 patients. Through GO and KEGG pathway analyses, 34 different proteins were found to be enriched in the spliceosome pathway. The expression levels of USP39 and STAT1 were significantly higher in HNSCC tumor tissue than in adjacent healthy tissue as assessed by LC-MS/MS analysis, and the increased expression of USP39 and STAT1 protein was confirmed by immunohistochemistry in clinical samples collected from 7 additional patients with HNSCC. Knockdown of USP39 or STAT1 inhibited the viability and migration of CAL27 and SCC25 cells. In addition, USP39 knockdown inhibited the expression of STAT1 mRNA in these cells. Conclusion: Our findings indicated that USP39 knockdown may inhibit HNSCC viability and migration by suppressing STAT1 expression. The results of this study suggest that USP39 may be a potential new target for HNSCC clinical therapy or a new biomarker for HNSCC.

Unveiling the potential of angiotensin-converting enzyme as a therapeutic target in head and neck squamous carcinoma

This study investigated the effects of ACE inhibition on Head and Neck Squamous Cell Carcinoma (HNSCC), tumor progression, and the relation of ACE2, AGTR1, ACE, and MAS1 expression with HNSCC patient` survival. ACE inhibitor (Captopril) treatment was applied into Swiss male mice. ACE2, AGTR1, ACE, and MAS1 expression levels were analyzed in HNSCC tumor tissues from The Cancer Genome Atlas (TCGA) database. ACE inhibition reduced cell viability, proliferation, migration, and invasion, increased ROS levels, and induced apoptosis in SCC9 cells. Neoplastic angiogenesis was suppressed by ACE inhibition in the CAM assay. ACE inhibition also reduced the incidence of severe dysplasia/carcinoma in the histological analysis. TCGA data revealed that lower ACE2 levels were associated with poorer survival outcomes, while increased AGTR1 expression correlated with a higher risk of death in HPV-negative patients. ACE upregulation and higher MAS1 expression were observed in HNSCC tumor tissues. ACE inhibition demonstrated anti-cancer effects in HNSCC cells and suppressed neoplastic angiogenesis. It also reduced histological aggressiveness and showed potential predictive value for ACE2 and AGTR1 expression levels in HNSCC. These findings suggest the therapeutic potential of ACE inhibitors in HNSCC treatment, requiring further investigation and validation in clinical settings.

SLA2 is a prognostic marker in HNSCC and correlates with immune cell infiltration in the tumor microenvironment

PurposeTo investigate Src-like adaptor 2 gene (SLA2) expression in head and neck squamous cell carcinoma (HNSCC), its potential prognostic value, and its effect on immune cell infiltration.MethodsThrough a variety of bioinformatics analyses, we extracted and analyzed data sets from the Cancer Genome Atlas (TCGA), Tumor Immune Estimation Resource (TIMER), and Gene Expression Profile Interaction Analysis (GEPIA) to analyze the correlation between SLA2 and the prognosis, immune checkpoint, tumor microenvironment (TME) and immune cell infiltration of HNSCC, and to explore its potential oncogenic mechanism. To further explore the potential role of SLA2 in HNSCC by Gene ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.ResultsSLA2 messenger ribonucleic acid (mRNA) levels were increased in HNSCC tumor tissues compared with normal tissues. In addition, we found that SLA2 may be an independent prognostic factor for HNSCC, and high SLA2 expression is associated with favorable prognosis in HNSCC. SLA2 expression was positively correlated with B cells, cluster of differentiation 8-positive T cells (CD8 + T cells), cluster of differentiation 4-positive T cells (CD4 + T cells), macrophages, neutrophil and dendritic cells infiltration. SLA2 has also been shown to co-express immune-related genes and immune checkpoints. Significant GO term analysis by Gene Set Enrichment Analysis (GSEA) indicated that genes correlated with SLA2 were located mainly in the side of membrane, receptor complex, secretory granule membrane, endocytic vesicle, membrane region, and endosome membrane, where they were involved in leukocyte cell–cell adhesion, response to interferon-gamma, and regulation of immune effector process. These related genes also served as antigen binding, cytokine receptor activity, phosphatidylinositol 3-kinase activity, peptide receptor activity, Src homology domain 3 (SH3) domain binding, and cytokine receptor binding. KEGG pathway analysis demonstrated that these genes related to SLA2 were mainly enriched in signal pathways, such as hematopoietic cell lineage, cell adhesion molecules (CAMs), natural killer cell mediated cytotoxicity, measles, and chemokine signaling pathway.ConclusionsSLA2 is increased in HNSCC, and high SLA2 expression is associated with favorable prognosis. SLA2 may affect tumor development by regulating tumor infiltrating cells in TME. SLA2 may be a potential target for immunotherapy.

Systematic review on oral microbial dysbiosis and its clinical associations with head and neck squamous cell carcinoma.

The relationship between head and neck squamous cell carcinoma (HNSCC) and the oral microbiome has been drawn in various studies. Microbial diversities, microbiome profiles, metagenomic analysis, and host-pathogen interactions were collected from these studies to highlight similarities and account for inconsistencies. We also evaluate the possible clinical applications of the microbiome regarding screening and diagnosis of HNSCC. Systematic analysis of studies regarding HNSCC and the microbiome was done according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement guidelines. Articles were retrieved from four databases (PubMed, ScienceDirect, CUHK Full-Text Journals, and Cochrane database) and were screened using predefined criteria. Twenty studies were chosen after screening for full-text review. α-diversity comparison was inconsistent whereas β-diversity between HNSCC and normal samples showed distinct clustering. Microbial dysbiosis characterized by change in the relative abundances of several bacterial species were also seen in HNSCC patients. At a phylum level, inconsistencies were seen between studies using HNSCC tumor tissue samples and saliva samples. At a genus level, Fusobacterium, Peptostreptococcus, Alloprevotella, Capnocytophaga, Catonella, and Prevotella were differentially enriched in HNSCC while Streptococcus, Actinomyces Veillonella, and Rothia were differentially depleted. Co-occurrence network analysis revealed a positive correlation of HNSCC with periodontal pathogens and a negative correlation with commensal bacteria. Metagenomic analysis of microbiota revealed a differential enrichment of pro-inflammatory genomic pathways which was consistent across various studies. Microbial dysbiosis was applied in clinical use as a tool for HNSCC screening. Random-forest analysis was adopted to differentiate between tumor and normal tissue, at 95.7% and 70.0% accuracies respectively in two studies. Microbial dysbiosis index was also used to predict prognosis. Oral microbial dysbiosis could be a promising tool for HNSCC screening and diagnosis. However, more research should be conducted pertaining to clinical applications to improve diagnostic accuracy and explore other clinical uses.

The lncRNA XIST/miR-29b-3p/COL3A1 axis regulates central carbon metabolism in head and neck squamous cell carcinoma and is associated with poor tumor prognosis.

Recent evidence shows that COL3A1 promotes the progression of many types of cancer. The purpose of our study is to explore the correlation between COL3A1 and the prognosis of patients with head and neck squamous cell carcinoma (HNSCC) and its potential mechanism. We initially screened the differentially expressed gene COL3A1 in The Cancer Genome Atlas (TCGA) database, and the association between the expression level of COL3A1, prognosis, and the clinical parameters of HNSCC patients was verified. A nomogram was constructed according to the multivariate analysis results. Next, a heatmap of COL3A1 co-expressed genes was constructed in TCGA database. The TargetScan database is used to explore the microRNAs (miRNA) related to COL3A1. The starBase database was used to explore and predict the long non-coding RNAs (lncRNAs) that the candidate miRNAs might bind to. Finally, the potential mechanism of action was investigated using Gene Set Enrichment Analysis (GSEA). COL3A1 expression is elevated in HNSCC tumor tissues, and HNSCC patients with high COL3A1 expression have worse prognostic factors. COL3A1 was positively correlated with the central carbon metabolism-related proteins: epidermal growth factor receptor (EGFR), phosphoglycerate mutase 1 (PGAM1), hexokinase 3 (HK3), and phosphofructokinase, platelet (PFKP). The TargetScan database showed that the best candidate miRNA for binding to the three prime untranslated region (3'UTR) end of COL3A1 mRNA was hsa-miR-29b-3p, which was negatively correlated with COL3A1. The starBase database showed that the lncRNA X Inactive Specific Transcript (lncRNA XIST) was the best candidate upstream non-coding RNA for regulating hsa-miR-29b-3p. GSEA showed that COL3A1 may be involved in the poor prognosis of HNSCC by participating in carbon metabolism, glucose metabolism, oxidative stress, and the Wingless-Type MMTV Integration Site Family (Wnt) and vascular endothelial growth factor A-vascular endothelial growth factor receptor 2 (VEGFA-VEGFR2) pathways. Low COL3A1 expression can be employed as a new HNSCC predictive biomarker, and the prognosis of HNSCC patients with lower COL3A1 expression can be greatly improved. At the same time, we found that the lncRNA XIST/miR-29b-3p/COL3A1 axis may regulate the central carbon metabolism of HNSCC and is associated with poor prognosis. These findings point to a potential target for developing HNSCC anticancer therapies.

Growth Regulated Oncogene-α Upregulates TNF-α and COX-2 and Activates NOD1/RIPK2 mediated-MAPK Pathway in Head and Neck Squamous Cell Carcinoma.

Purpose: The long-term prognosis and survival rate of patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) are poor, although the identification of specific biomarkers that reveal its nature and aggressiveness has improved it. Growth-related oncogene alpha (Groα) and NOD1 (nucleotide-binding oligomerization domain 1) can be used as prognosis markers to identify subgroups of HNSCC patients with low survival rates and as potential therapeutic targets for HNSCC patients. However, the mechanism associated with the Groα-mediated NOD pathway in HNSCC progression remains unclear. Method: Overall survival analysis and multiple-gene comparison were analyzed using Gene Expression Profiling Interactive Analysis (GEPIA). qRT-PCR and RT-PCR were used to analyze mRNA expression. Microarray, immunofluorescence staining or western blot analyses were carried out to detect protein expression. Results: Groα was significantly higher in the grade 4 HNSCC tumor tissues compared with that in grade 1-3 and healthy subjects. High expression of Groα, NOD1 and RIPK2 (receptor-interacting serine-threonine kinase 2) is correlated with survival rate in HNSCC patients. Treatment of SCC25 and OECM-1 cells with Groα increased the expression of NOD1 and RIPK2 in a concentration-dependent manner. The findings herein reveal the association of Groα, NOD1 and RIPK2 biomarkers with HNSCC carcinogenesis. Moreover, Groα is the major stimulus of inflammatory mediation and promotes TNF-α (tumor necrosis factor-α) and COX-2 (cyclooxygenase-2) expression in HNSCC. Groα induces TNF-α and COX-2 expression through regulation involving ERK (extracellular signal-regulated kinase)-, JNK (C-Jun N-terminal kinase)- and p38 MAPK (mitogen-activated protein kinase)-dependent signaling pathways. Conclusions: Our findings herein constitute the first evidence that Groα is important in HNSCC progression and metastasis via the NOD1-mediated MAPK pathway, suggesting a role for Groα and NOD1 in mediating metastasis and its potential as a therapeutic target.

YTHDC1 Promotes Stemness Maintenance and Malignant Progression in Head and Neck Squamous Cell Carcinoma.

YTH domain containing 1 (YTHDC1), an N6-methyladenosine (m6A) modification reading protein, plays a key role in regulating RNA translation and degradation. However, the role of YTHDC1 in head and neck squamous cell carcinoma (HNSCC) cancer stem cells remains largely unknown. This study is aimed at investigating the role of YTHDC1 in HNSCC and exploring its role in regulating cancer stem cells. RNA sequencing was used to detect differentially expressed genes (DEGs) between SCC9 spheres and SCC9 cells and to uncover molecular pathways and target molecules associated with CSCs. We detected YTHDC1 expression in The Cancer Genome Atlas (TCGA) database data and clinical samples. Subsequently, YTHDC1 gene suppression assays were performed in HNSCC cell lines to investigate the effect of YTHDC1 on tumor cell stemness maintenance, proliferation, and migration capacity. To further confirm the role of YTHDC1 in regulating cancer stem cells in HNSCC, we analyzed online HNSCC single-cell transcriptomic data to investigate YTHDC1 expression patterns at the single-cell level and the correlation of these levels with the expression of stem cell markers. YTHDC1 expression levels were significantly upregulated in SCC9 spheres, and YTHDC1 was aberrantly expressed in HNSCC tumor tissues. The increased YTHDC1 expression was closely correlated with the clinical characteristics of HNSCC patients. YTHDC1 regulates the malignant phenotype of HNSCC in both in vivo and in vitro studies. Further single-cell transcriptomic data analysis revealed that YTHDC1 positively correlated with malignant epithelial cell stemness capacity at the single-cell level, and that YTHDC1 was involved in regulating stemness maintenance in HNSCC. These findings suggest that YTHDC1 may serve as a biomarker for stem maintenance and malignant progression in HNSCC, providing new insights into the treatment of cancer.

Dickkopf-1 is a Prognostic Biomarker of Head and Neck Squamous Cell Carcinoma that Correlates with Immune Infiltrates

<h3>Purpose/Objective(s)</h3> Head and neck squamous cell carcinoma (HNSCC) is associated with high immune cell infiltration, and immunotherapy is currently one of the most promising treatments for HNSCC. Dickkopf-1 (DKK1), a Wnt-signaling inhibitor and mediator of immune activation, is highly associated with tumor progression and the tumor microenvironment and, thus, disease prognosis. However, the associations between DKK1 expression, HNSCC prognosis, and tumor-infiltrating lymphocytes remain unknown. <h3>Materials/Methods</h3> We investigated these relationships in this study by exploring DKK1-expression differences in multiple tumor tissues and normal tissues, using the TIMER, Oncomine, and UALCAN databases. We searched the Kaplan–Meier database to assess correlations between DKK1 mRNA-expression levels and clinical outcomes. Subsequently, we evaluate the correlation between DKK1 expression and tumor immune-cell infiltration using the TIMER platform and TISIDB website. In addition, we assessed DKK1, CD3, and CD4 expression in 27 head and neck tumor samples and DKK1 expression in 15 normal head and neck tissue samples by immunohistochemistry. Moreover, we used the cBioPortal and UCSC Xena databases to explore aberrant DKK1 expression during tumorigenesis. <h3>Results</h3> DKK1 significantly upregulated in HNSCC tissues versus normal tissues. DKK1 expression was related to the HNSCC tumor stage, tumor grade, histology, lymph node metastasis, and a poor clinical prognosis. DKK1 expression correlated negatively with CD3+ (P < 0.0001) and CD4+ (P < 0.0001) immune cell infiltration in HNSCC tumor tissues. Moreover, DKK1 closely correlated with genetic markers of various immune cells. The prognosis of HNSCC correlated negatively with DKK1 expression in immune cells. Copy-number variation and DNA methylation may promote abnormal DKK1 upregulation in HNSCC. These results help understand the role of DKK1 in HNSCC immunotherapy and provide a rationale for clinical research on treating HNSCC by targeting DKK1. <h3>Conclusion</h3> DKK1 may therefore serve as a prognostic indicator and a biomarker for immune infiltration in HNSCC, and potentially as a central target for improving the diagnosis and immunotherapy efficacy of HNSCC.

CircMAT2B facilitates the progression of head and neck squamous cell carcinoma via sponging miR-491-5p to trigger ASCT2-mediated glutaminolysis.

Circular RNAs (circRNAs) are well-known to exert significant roles in regulating the pathological processes, including human carcinogenesis. Currently, less is known about their exact roles in head and neck squamous cell carcinoma (HNSCC). Herein, we aimed to investigate and validate the role of a novel circRNA, circMAT2B, as well as its potential molecular mechanism in HNSCC progression. A cohort of 41 paired of HNSCC tumor tissues and adjacent normal tissues from HNSCC patients were collected. Further, we characterized circMAT2B expression patterns in HNSCC tissues and cell lines, as well as exploring its association with the prognosis of HNSCC patients. Biological functions on cell proliferation, apoptosis, migration, and invasion were assessed using Cell Counting Kit-8, EdU incorporation, TUNEL, wound healing, and transwell assays. Glutaminolysis was evaluated by measuring glutamine, glutamate, and α-ketoglutarate (α-KG) levels. The regulatory network of circMAT2B/miR-491-5p/ASCT2 axis was verified by RNA immunoprecipitation and luciferase reporter assays. Western blot was conducted to detect the level of ASCT2 and GLS1. Remarkably overexpressed circMAT2B was observed in HNSCC tissues and cell lines, of which high abundance was positively correlated with patients' poor prognosis. Silencing of circMAT2B inhibited cell proliferation, migration, and invasion, as well as glutaminolysis. miR-491-5p, interacted with ASCT2, was identified to be a downstream target of circMAT2B, thereby involving in circMAT2B-mediated biological effects. In summary, we draw a conclusion that circMAT2B could modulate the processes of cell proliferation, migration, invasion, and glutaminolysis of HNSCC cells partly via the miR-491-5p/ASCT2 axis by a molecular mechanism of competing endogenous RNA (ceRNA), implying an underlying circRNA-targeted therapy for HNSCC treatment.

A Four-Gene Signature Associated with Radioresistance in Head and Neck Squamous Cell Carcinoma Identified by Text Mining and Data Analysis.

Purpose Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cancer globally, and radiotherapy plays a crucial part in its treatment. This study was designed to identify potential genes related to radiation resistance in HNSCC. Method We first used text mining to obtain common genes related to radiotherapy resistance and HNSCC in published articles. Functional enrichment analyses were conducted to identify the significantly enriched pathways and genes. Protein and protein interactions were performed, and the most significant gene modules were determined; then, genes in the gene modules were validated at transcriptional levels and overall survival. Gene set variation analysis (GSVA) score was calculated, and the association between GSVA score and survival/pathway was estimated. Immune cell infiltration, methylation, and genetic alteration analysis of these genes was conducted in HNSCC patients. Finally, potential sensitive anticancer drugs related to target genes were obtained. Result We identified 583 common genes through text mining. After further validation, a four-gene signature (EPHB2, SPP1, SERPINE1, and VEGFC) was constructed. The patients with higher GSVA scores have a worse prognosis than those with lower GSVA scores. Differences in methylation of these four genes in HNSCC tumor tissue and normal tissue were compared, with higher methylation levels of EBPH2 and SPP1 in normal tissue and higher methylation levels of SERPINE1 in the tumor. Immune cell infiltration revealed that the increased expression of these genes was closely related to the infiltration level of CD4+ T cell, neutrophil, macrophage, and dendritic cell. Thirty drugs, including 22 positively and eight negatively correlated drugs that most correlated with related genes, were available for treating HNSCC. Conclusion In this study, we identified four potential genes as well as corresponding drugs that might be related to radioresistance in HNSCC patients. These candidate genes may provide a promising avenue to further elevate radiotherapy efficacy.

CCR5 as a prognostic biomarker correlated with immune infiltrates in head and neck squamous cell carcinoma by bioinformatic study

BackgroundC-C chemokine receptor 5 (CCR5) has recently been recognized as an underlying therapeutic target for various malignancies. However, the association of CCR5 with prognosis in the head and neck squamous cell carcinoma (HNSC) patients and tumor-infiltrating lymphocytes (TILs) is unclear.MethodsIn the current experiment, methods such as the Tumor Immune Estimation Resource Analysis (TIMER), Gene Expression Profiling Interactive Analysis (GEPIA), UALCAN, and Kaplan-Meier plotter Analysis were used to comprehensively evaluate the expression of CCR5 in human various malignancies and the clinical prognosis in HNSC patients. Subsequently, we used the TIMER database and the TISIDB platform to investigate the correlation between CCR5 expression levels and immune cell infiltration in the HNSC tumor microenvironment. Furthermore, immunomodulatory and chemokine profiling were performed using the TISIDB platform to analyse the correlation between CCR5 expression levels and immunomodulation in HNSC patients.ResultsWe found that CCR5 expression in HNSC tumor tissues was significantly upregulated than in normal tissues. In HNSC, patients with high CCR5 expression levels had worse overall survival (OS, HR = 0.59, p = 0.00015) and worse recurrence-free survival (RFS, HR = 3.27, p = 0.00098). Upregulation of CCR5 expression is closely associated with immunomodulators, chemokines, and infiltrating levels of CD4+ T cells, neutrophils, macrophages, and myeloid dendritic cells. Furthermore, upregulated CCR5 was significantly associated with different immune markers in the immune cell subsets of HNSC.ConclusionsHigh expression of CCR5 plays an important prognostic role in HNSC patients and may serve as a prognostic biomarker correlated with immune infiltration, and further studies are still needed to investigate therapeutic targeting HNSC patients in the future.

E2F4 transcription factor is a prognostic biomarker related to immune infiltration of head and neck squamous cell carcinoma

To investigate the relationship between the transcription factor, E2F4, and head and neck squamous cell carcinoma (HNSCC), and to preliminarily explore the signaling pathways and immunological role of E2F4. The mRNA expression of E2F4 in HNSCC was evaluated by searching Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) datasets. E2F4 protein expression was analyzed by immunohistochemistry using the CMU1h-ENT database. The association between E2F4 expression and tumor infiltration of immune cells was analyzed. Intracellular signaling by E2F4 was explored using KEGG and GO analysis. The correlation of E2F4 expression with clinical characteristics and its prognostic role were validated and analyzed in TCGA database. From the analysis of GEO and TCGA data, E2F4 expression was found to be up-regulated in HNSCC tumor tissues, and its level was associated with T, Grade, and M staging. Kaplan–Meier curve and Cox analyses indicated that the high expression of E2F4 was related to a poor prognosis. Thus, E2F4 was considered a potential prognostic factor for HNSCC. Immunohistochemical staining showed that E2F4 was mainly localized in the cell nucleus; it was highly expressed in HNSCC tissues, with a significant difference noted from that in pericancerous mucosa tissues. A correlation was observed between the differential expression of E2F4 and the immune infiltration of HNSCC. As revealed by KEGG and GO analysis, differential enrichment was found in the cell cycle, spliceosome, meiosis, microbial polysaccharide synthesis, and WNT signaling pathway, as well as in cyclic adenosine monophosphate, ERBB2, VEGF, GCNP and MYC pathways. E2F4 plays an important role in tumor progression and may be a critical biological prognostic factor for HNSCC. In addition, it functions in the nucleus as a transcription factor, regulates immune cells, and could be a promising molecular target for the diagnosis and treatment of HNSCC.

Abstract 2959: Investigating the oncogenic effects and mechanisms of the SMYD3 methyltransferase in HPV-negative head and neck squamous cell carcinoma

Abstract Human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer type in the world and is associated with an overall poor prognosis. The protein methyltransferase SET and MYND domain-containing 3 (SMYD3), which trimethylates H3K4 and H4K20, can stimulate gene transcription and activate several oncogenic pathways, including epithelial-mesenchymal transition, and cell cycle related pathways. Additionally, its expression is associated with a poor prognosis in various cancer types, such as colon and ovarian cancer. This study aims to decipher whether SMYD3 has direct oncogenic effects and relevant mechanisms in HPV-negative HNSCC. Our preliminary results have demonstrated that SMYD3 is significantly overexpressed in HPV-negative HNSCC compared to normal and dysplastic epithelium. SMYD3 knockdown decreased cellular proliferation and clonal capacity in CFAs after siRNA-mediated SMYD3 knockdown in several HPV-negative HNSCC cell lines or CRISPR-mediated SMYD3 knockout. Cell cycle analysis of CRISPR SMYD3 KO cell lines showed a significant decrease in the S phase, supporting that cell cycle arrest may be a mechanism through which this decreased cellular proliferation occurs. Nuclear/cytoplasmic dissociation and western blotting of 6 HPV-negative HNSCC cell lines, and immunohistochemistry for SMYD3 in primary and recurrent/metastatic HPV-negative HNSCC tumor tissues have identified the expression of SMYD3 in both the cytoplasm and nucleus, with greater expression in the cytoplasm than the nucleus. Ongoing experiments aim to investigate the effects of SMYD3 knockdown on the global proteome, as well as its effects on histone post-translational modifications in HPV-negative HNSCC cell lines. Genome-wide mapping for SMYD3, H3K4me3 and H4K20me3 in HPV- negative HNSCC cells using CUT&amp;RUN assays are ongoing with the goal to identify direct downstream gene targets regulated by human SMYD3. Finally, we plan to investigate if SMYD3 knockout decreases tumor growth in NSG xenografts. This study may provide the biological rational to target SMYD3 as a novel therapeutic avenue in HPV-negative HNSCC. Citation Format: Madhavi Murali, Daniel Tsai, Nupur Nigam, Kyunghee Burkitt, Sohyoung Kim, Hui Cheng, Vassiliki Saloura. Investigating the oncogenic effects and mechanisms of the SMYD3 methyltransferase in HPV-negative head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2959.

Sensory Neurotransmitter CGRP Modulates Oral Cancer Growth and Cancer-associated Immune Response

Head and neck squamous cell carcinoma (HNSCC) induces severe pain due in part to activation of primary afferent neurons by cancer-secreted mediators. Local neurotransmitter release (e.g., calcitonin gene-related peptide (CGRP)) from trigeminal neurons innervating the cancer has been linked to tumorigenesis. We hypothesize that CGRP exerts a dual effect on both cancer-associated pain and tumor progression, suggesting that CGRP may be a promising therapeutic target in HNSCC treatment. We used human tumor tissue and patient-reported outcomes to explore the relationship between CGRP+ sensory nerve innervation and cancer pain in patients. To determine CGRP receptor expression on tumor cells, immunohistochemistry and PCR were performed on human and mouse oral cancer cell lines. We used a syngeneic tongue tumor transplant mouse model of oral cancer and a global Calca knockout mouse (i.e. CGRP-KO) to investigate the impact of CGRP signaling on tumor growth and the associate immune response in vivo. We found prominent CGRP-immunoreactive sensory nerve presence innervating human HNSCC tumor tissue, which positively correlated to patient-reported pain (r2=0.357). Furthermore, human HNSCC cell lines expressed 3-fold more CGRP receptor, RAMP1, compared to a non-tumorigenic keratinocyte cell line. In tumor-bearing CGRP-KO mice, we found a significant reduction in tumor size at post-inoculation days 7 and 14 compared to wildtype. We also found a 4-fold increase in tumor infiltrating RAMP1-expressing CD4+ T cells, as well as a 5-fold increase cytotoxic CD8+ T cells and NK1.1+ NK cells in tumor tissue CGRP-KO mice compared to wildtype. This preliminary data suggests that CGRP signaling from sensory neurons may increase cancer associated pain and tumor progression. Further knowledge regarding the relationship between sensory neurons and cancer could allow for the repurposing clinically available nervous system drugs (e.g., anti-CGRP antibodies) for the treatment of cancer and cancer pain. Grant support from the Rita Allen Foundation.

Preclinical evidence for pioglitazone and bexarotene combination in oral cancer chemoprevention.

Head and neck squamous cell carcinoma (HNSCC) requires new treatments and targeted approaches to improve survival. The peroxisome proliferator-activated receptor γ (PPARγ) and retinoic X receptor alpha (RXRα) nuclear receptor pathways may be targetable with repurposed Food and Drug Administration (FDA)-approved agents for prevention and treatment. Oral cancer and leukoplakia cell lines were treated with the PPARγ agonist (pioglitazone) and RXRα activator (bexarotene). PPARγ activation, cellular proliferation, apoptosis activity and phenotype, including the pharmacodynamic marker, involucrin (IVL), were subsequently analyzed using a reporter gene assay, genomic data, MTT assay and western blot. Microarray analysis of HNSCC tumor versus normal tissue shows IVL expression is significantly increased in normal tissue compared to HNSCC tumors (p < 0.0001). In MSK Leuk1 and CA 9-22 cell lines, pioglitazone increases PPARγ DNA binding activity and IVL promoter activity in a dose dependent manner (p < 0.01 and p < 0.0001). Combination treatment with pioglitazone and bexarotene increases PPARγ DNA binding activity and IVL promoter activity (p < 0.01 and p < 0.0001). MTT analysis shows decreases in cell proliferation when cells are treated with pioglitazone and bexarotene. Decreases in cell proliferation are significant to at least p < 0.05 for all combination versus single agent treatments. Western blot on whole-cell lysate from cells treated with pioglitazone and bexarotene alone or in combination for IVL showed increased protein levels with combination treatment. Targeting the PPARγ/RXRα heterodimer with pioglitazone and bexarotene was effective in this preclinical project. This was functional in both preneoplastic and oral cancer cell lines. A better understanding of the molecular mechanism on downstream effects on cellular proliferation could potentially have implications clinically, both in oral preneoplasia and possibly head and neck cancer; however, more research needs to be done to explore the potential these medications have in chemoprevention.

The Immune Infiltration in HNSCC and Its Clinical Value: A Comprehensive Study Based on the TCGA and GEO Databases.

Background Being potential field of research for tumor immunological therapy, the head and neck squamous cell carcinoma (HNSCC) is one of most discussed types of tumor. Recently, some clinical trials have also used immunological therapy and demonstrated a subset of HNSCC patients who have shown a clear longer survival time. Objective To conduct further studies and deeper research in the immunological oncology of HNSCC, a more detailed description and comprehending of the complicated landscape of immune infiltrative may be required. Methods Firstly, we have described the fraction of different infiltrating immune cells in the HNSCC tumor and then compared it to the normal tissue, and secondly, we have explored the clinical implications of various infiltrated immune cell fractions meticulously. The gene expression profiles of HNSCC tissue were obtained from databases of TCGA and GEO and utilized the deconvolution algorithm (CIBERSORT) to presume the fractions of 22 several immune sensitive cells. Results Our results indicated that the immune infiltrating cell fractions were considerably different between HNSCC tumor tissue and paired normal tissue, but at the same time, we found a potential internal correlation among the immune cells and also showed the association between immune infiltrating cells and their clinical characteristics. It is worth noting that the resting dendritic cells and M1 macrophages were linked with a favorable prognosis, while the CD4+ T cells with a poorer outcome. Conclusion Fractions of immune cell percentage were also associated with tumors' pathological grade, age, and TNM stage.

Genetic variants of cell cycle pathway genes are associated with head and neck squamous cell carcinoma in the Chinese population.

Genetic alterations in the cell cycle pathway are common in head and neck squamous cell carcinoma (HNSCC). We identified four novel HNSCC susceptibility loci (CDKN1C rs452338, CDK4 rs2072052, E2F2 rs3820028 and E2F2 rs2075993) through a two-stage matched case-control study. There was a combined effect among the four single nucleotide polymorphisms (SNPs), as the number of risk genotypes increased, the risk of HNSCC displayed an increasing trend (Ptrend < 0.001). And there were multiplicative interactions between rs452338 and rs2072052, rs2072052 and rs3820028, rs2072052 and rs2075993. Functional bioinformatics analysis and dual-luciferase reporter assay revealed that E2F2 rs2075993 T>C reduced the stability of E2F2 3'-UTR secondary structure and affected the binding of E2F2 to miR-940, which was up-regulated in HNSCC tumor tissues (P = 2.9e-8) and was correlated with poor overall survival of HNSCC (HR = 1.39, 95% CI = 1.02-1.90). In vitro assays, we discovered that the expression of miR-940 was regulated by METTL3, and miR-940 promoted the proliferation, migration and invasion, and inhibited the senescence and autophagy of tumor cells. In terms of mechanism, compared with rs2075993 allele T, we found that the protective variant rs2075993 allele C interfered with the translational inhibition of E2F2 by miR-940, resulting in increased expression of E2F2 protein, which further reduced the proliferation, migration, invasion, and increased the senescence of tumor cells.

SEC61G identified as a prognostic biomarker of head and neck squamous cell carcinoma

PurposeIt is of obvious interest to identify clinical prognosis-related oncogenes in HNSCC (head and neck squamous cell carcinoma).MethodsBased on the available datasets within the TCGA (The Cancer Genome Atlas) and the GEO (Gene Expression Omnibus) databases, the potential mechanism of action of the SEC61G (SEC61 translocon subunit gamma) gene in HNSCC tumorigenesis was explored by several bioinformatics approaches.ResultsThere was a higher expression level of SEC61G in primary HNSCC tumor tissues than in normal tissues. Moreover, highly expressed SEC61G was statistically associated with the poor survival prognosis of HNSCC patients. When HPV (human papilloma virus) was considered, we also observed a relatively lower proportion of “arm-level gain” and “high amplification” types of CNA (copy-number alteration) in the HNSCC-HPV (+) group than in the HNSCC-HPV (−) group. Additionally, we identified SEC61G CAN-correlated genes, such as CCT6A (chaperonin-containing TCP1 subunit 6A) and HUS1 (HUS1 checkpoint clamp component), and found a correlation between SEC61G copy-number segments and prognosis related to overall and progression-free survival intervals of HNSCC patients. Moreover, the molecular regulation mechanisms of the spliceosome, ribosome, proteasome degradation, cell adhesion, and immune infiltration of B and CD8+ T cells may contribute to the involvement of SEC61G in the pathogenesis of HNSCC.ConclusionsThe SEC61G gene was identified for the first time as a prognostic biomarker of HNSCC. The detailed underlying mechanism merits further research.