HNSCC Tissues Research Articles

ITGA5 is associated with prognosis marker and immunosuppression in head and neck squamous cell carcinoma

BackgroundHead and neck squamous cell carcinoma (HNSCC) is a major tumor that seriously threatens the health of the head and neck or mucosal system. It is manifested as a malignant phenotype of high metastasis and invasion caused by squamous cell transformation in the tissue area. Therefore, it is necessary to search for a biomarker that can systematically correlate and reflect the prognosis of HNSCC based on the characteristics of head and neck tumors.MethodsBased on TCGA-HNSCC data, R software was used to analyze gene expression, correlation, Venn diagram, immune invasive and immunosuppressive phenotypes respectively. The intrinsic effect of ITGA5 on the malignant phenotype of HNSCC cells was verified by cell experiments. Immunohistochemical images from The Human Protein Atlas (THPA) database display the differences in the expression of related proteins in HNSCC tissues. Based on functional enrichment and correlation analysis, the prognostic value of ITGA5 for HNSCC was explored, and the expression level of ITGA5 may affect the chemotherapy of targeting the PI3K-AKT.ResultsIn this study, the target gene ITGA5 may be identified as a valuable prognostic marker for HNSCC. The results of enrichment analysis showed that ITGA5 was mainly involved in the dynamic process of extracellular matrix, which may affect the migration or metastasis of tumor cells. Meanwhile, ITGA5 may be closely related to the infiltration of M2 macrophages, and its secretory phenotypes TGFB1, PDGFA and PDGFB may affect the immunosuppressive phenotypes of tumor cells, which reflects the systemic influence of ITGA5 in HNSCC. In addition, the expression levels of ITGA5 were negatively correlated with the efficacy of targeting PI3K-AKT chemotherapy.ConclusionITGA5 can be used as a potential marker to systematically associate with prognosis of HNSCC, which may be associated with HNSCC malignant phenotype, immunosuppression and chemotherapy resistance.

Universal penalized regression (Elastic-net) model with differentially methylated promoters for oral cancer prediction

BackgroundDNA methylation showed notable potential to act as a diagnostic marker in many cancers. Many studies proposed DNA methylation biomarker in OSCC detection, while most of these studies are limited to specific cohorts or geographical location. However, the generalizability of DNA methylation as a diagnostic marker in oral cancer across different geographical locations is yet to be investigated.MethodsWe used genome-wide methylation data from 384 oral cavity cancer and normal tissues from TCGA HNSCC and eastern India. The common differentially methylated CpGs in these two cohorts were used to develop an Elastic-net model that can be used for the diagnosis of OSCC. The model was validated using 812 HNSCC and normal samples from different anatomical sites of oral cavity from seven countries. Droplet Digital PCR of methyl-sensitive restriction enzyme digested DNA (ddMSRE) was used for quantification of methylation and validation of the model with 22 OSCC and 22 contralateral normal samples. Additionally, pyrosequencing was used to validate the model using 46 OSCC and 25 adjacent normal and 21 contralateral normal tissue samples.ResultsWith ddMSRE, our model showed 91% sensitivity, 100% specificity, and 95% accuracy in classifying OSCC from the contralateral normal tissues. Validation of the model with pyrosequencing also showed 96% sensitivity, 91% specificity, and 93% accuracy for classifying the OSCC from contralateral normal samples, while in case of adjacent normal samples we found similar sensitivity but with 20% specificity, suggesting the presence of early disease methylation signature at the adjacent normal samples. Methylation array data of HNSCC and normal tissues from different geographical locations and different anatomical sites showed comparable sensitivity, specificity, and accuracy in detecting oral cavity cancer with across. Similar results were also observed for different stages of oral cavity cancer.ConclusionsOur model identified crucial genomic regions affected by DNA methylation in OSCC and showed similar accuracy in detecting oral cancer across different geographical locations. The high specificity of this model in classifying contralateral normal samples from the oral cancer compared to the adjacent normal samples suggested applicability of the model in early detection.

MRE11 as a plausible biomarker and prognostic bioindicator for head and neck squamous cell carcinoma

ObjectiveHead and Neck Squamous Cell Carcinoma (HNSCC) ranks as the sixth most prevalent form of cancer worldwide. MRE11 protein contains multiple domains that play a role in the initiation of DNA repair. This study aimed to elucidate the expression and prognostic significance of MRE11 in HNSCC. Material and methodsThe Cancer Genome Atlas (TCGA-HNSCC) dataset comprising 520 HNSCC tissues and 44 normal tissues was initially used to evaluate the association between MRE11 expression and clinicopathologic characteristics. Kaplan–Meier plot was utilized for survival analysis. MRE11-immune cell interaction was analyzed using Tumor Immune Estimation Resource (TIMER) database. Further, Insilco methods were used to explore the protein network and its association with other pathways. Quantitative reverse transcription PCR (RT-qPCR) was used to validate the MRE11 mRNA expression in oral squamous cell carcinoma (OSCC) tissues in patient samples. ResultsMRE11 expression was upregulated in HNSCC, and the expression significantly varied across different clinical stages, pathological grades, and initial treatment outcomes. Further, high MRE11 expression is associated with poorer survival outcomes. MRE11 overexpression is also linked to the activation of the HIPPO signaling pathway, the mTOR signaling pathway, and the MYC/MYCN signaling pathway. ConclusionMRE 11 can be considered a novel prognostic biomarker for HNSCC, which can be leveraged for promising treatment outcomes. This research highlights MRE11 as a novel molecular biomarker for HNSCC and offers a new direction for its treatment, explicitly targeting MRE11 and its network for therapeutic intervention.

GINS2 promotes the progression of human HNSCC by altering RRM2 expression.

GINS2 exerts a carcinogenic effect in multiple human malignancies, while it is still unclear that the potential roles and underlying mechanisms of GINS2 in HNSCC. TCGA database was used to screen out genes with significant differences in expression in HNSCC. Immunohistochemistry and qRT-PCR were used to measure the expression of GINS2 in HNSCC tissues and cells. GINS2 was detected by qRT-PCR or western blot after knockdown or overexpression. Celigo cell counting, MTT, colony formation, and flow cytometric assay were used to check the ability of proliferation and apoptosis. Bioinformatics and microarray were used to screen out the downstream genes of GINS2. GINS2 in HNSCC tissues and cells was up-regulated, which was correlated with poor prognosis. GINS2 gene expression was successfully inhibited and overexpressed in HNSCC cells. Knockdown of GINS2 could inhibit proliferation and increase apoptosis of cells. Meanwhile, overexpression of GINS2 could enhance cell proliferation and colony formation. Knockdown of RRM2 may inhibit HNSCC cell proliferation, while overexpression of RRM2 rescued the effect of reducing GINS2 expression. Our study reported the role of GINS2 in HNSCC for the first time. The results demonstrated that in HNSCC cells, GINS2 promoted proliferation and inhibited apoptosis via altering RRM2 expression. Therefore, GINS2 might play a carcinogen in HNSCC, and become a specific promising therapeutic target.

Pathogenic loss-of-function mutations in LRP1B are associated with poor survival in head and neck cancer patients

ObjectiveHead and neck squamous cell carcinoma (HNSCC) present a significant challenge in the medical field due to treatment resistance, which often hinders successful outcomes. The dysregulation of the LRP1B gene is linked to various cancers, but its specific role in HNSCC is poorly understood. MethodsThis study investigated the link between pathogenic loss-of-function mutations in the LRP1B gene and survival outcomes in HNSCC patients. The Cancer Genome Atlas HNSCC cohort, comprised of 520 tumor and 44 normal tissues, was analyzed using cBioportal, and UALCAN tools. Expression patterns, survival outcomes, and clinical correlations of LRP1B were evaluated. In-depth analyses involved validation of mRNA expression using RT-qPCR and functional exploration using various in-silico tools. ResultsAnalysis of data from The Cancer Genome Atlas (TCGA) and cBioPortal revealed a high frequency (25 %) of LRP1B mutations in HNSCC patients. Notably, splice mutation, truncating mutation, and deep deletion, considered potential drivers, are commonly associated with LRP1B mutations. Patients with LRP1B mutations also exhibit poorer overall survival rates compared to those without these mutations. Furthermore, LRP1B mRNA expression is significantly reduced in HNSCC tissues compared to normal tissues and is correlated with advanced tumor stage, higher tumor grade, and nodal metastasis. ConclusionThese findings indicate that LRP1B may function as both a prognostic biomarker and a therapeutic target in HNSCC patients.

Increased SEC14L2 expression is associated with clinicopathological features and worse prognosis in oral squamous cell carcinoma.

Abnormal expression of SEC14L2 has been implicated in many human cancers. However, the role of SEC14L2 in oral squamous cell carcinoma (OSCC) remains unclear. Therefore, this study aimed to evaluate the expression and prognostic roles of SEC14L2 in OSCC. OSCC tumors and adjacent non-tumors were collected from OSCC patients and used for SEC14L2 mRNA expression by quantitative reverse transcription PCR (RT-qPCR). Additionally, the expression of SEC14L2 was further analyzed using The Cancer Genome Atlas-Head Neck Squamous Cell Carcinoma (TCGA-HNSCC) dataset to identify its relationship with HNSCC clinical characteristics. The Kaplan-Meier plot was used to assess survival rates, and the Tumor Immune Estimation Resource (TIMER) database was used to examine the correlation between SEC14L2 expression and tumor immune cell infiltration. In silico tools also looked at SEC14L2 involvement in cancer pathways through its protein network. The mRNA and protein levels of SEC14L2 are notably higher in both OSCC and HNSCC tissues compared to adjacent normal tissues. Upregulation of SEC14L2 was associated with advanced tumor stages, grades, metastasis, HPV-negative, and TP53 mutations in cancer patients. In addition, the high expression of SEC14L2 was negatively correlated with the poor survival of cancer patients and the infiltration of diverse immune cells in cancer patients. According to the findings of this investigation, SEC14L2 is significantly elevated in OSCC/HNSCC patients and associated with a worse prognosis. More investigation and clinical studies are required to completely understand the therapeutic potential of SEC14L2 in HNSCC and convert these findings into better patient outcomes.

Abstract 5188: Defining PD-1/PD-L1 receptor-ligand interactions in the head and neck cancer tumor microenvironment identifies unique ‘immune cell rivers’ of cellular interactions

Abstract Head and neck squamous cell carcinoma (HNSCC) is the 7th most common cancer globally with a poor 5-year survival rate of ~50%. With the emerging successes with immune checkpoint blockade (ICB) in the metastatic and recurrent setting, there is a need for predictive biomarkers to identify patients likely to achieve clinical benefit. Whilst PD-L1 expression on tumour cells and PD-1 on immune cells have been investigated, these remain inconclusive biomarkers. To better understand and map the PD-L1/PD-1 receptor-ligand interactions, we evaluated a novel in situ proximity ligation assay (is PLA) across whole tissue sections of pre-treatment HNSCC patients to received ICB therapy. Pre-treatment FFPE whole tissue samples were collected from n=25 advanced stage HNSCC patients across two major Queensland Hospitals (Royal Brisbane and Women’s Hospital and the Princess Alexandra Hospital). Tissues were stained with multiplex immunofluorescence and the Navinci Diagnostics fluorescent is PLA assay for PD-1/PD-L1. Tissues were incubated with the PD1/PD-L1 antibodies and subsequent incubation with oligonucleotide-tagged secondary probes enabled PLA, which highlights PD1 and PD-L1 interactions. Following the application of secondary probes, the sections underwent a ligation step, then a post-blocking, and an amplification process to enhance the detection of protein interactions. Next, the sections were treated with a detection solution to visualize the amplification product. The PLA assay findings were measured against clinical endpoints (PFS/OS criteria). In n=5/25 HNSCC tissues, positive PD-1/PD-L1 interactions were measured across the tumour microenvironment (TME), specifically in the immune rivers on the immediate tumour periphery. These cells showed highly specific binding of the PLA product. These areas positive by the PLA assay were in stark contrast to the tumour bulk/nests which were absent in PD-1/PD-L1 interactions. Notably, the PLA PD-1/PD-L1 do not correlate with tumour PD-L1 expression or tumour proportion scores (TPS). Rather, our study highlighted the difference in spatially mapping PD-1/PD-L1 interactions which may better recapitulate the tumour-immune recognition and activity in the TME. Taken together, these data provide a novel approach for determining clinically relevant receptor/ligand interactions in the HNSCC TME. Citation Format: Habib Sadeghirad, Vahid Yaghoubi Naei, James Monkman, Subham Basu, Agata Wicher, Rahul Ladwa, Brett GM Hughes, Arutha Kulasinghe. Defining PD-1/PD-L1 receptor-ligand interactions in the head and neck cancer tumor microenvironment identifies unique ‘immune cell rivers’ of cellular interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5188.

KLF7 regulates super-enhancer-driven IGF2BP2 overexpression to promote the progression of head and neck squamous cell carcinoma

BackgroundHead and neck squamous carcinoma (HNSCC) is known for its high aggressiveness and susceptibility to cervical lymph node metastasis, which greatly contributes to its poor prognosis. During tumorigenesis, many types of cancer cells acquire oncogenic super-enhancers (SEs) that drive the overexpression of oncogenes, thereby maintaining malignant progression. This study aimed to identify and validate the role of oncogenic SE-associated genes in the malignant progression of HNSCC.MethodsWe identified HNSCC cell-specific SE-associated genes through H3K27Ac ChIP-seq and overlapped them with HNSCC-associated genes obtained from The Cancer Genome Atlas (TCGA) dataset and Gene Expression Omnibus (GEO) datasets using weighted gene coexpression network analysis (WGCNA) to identify hub genes. The expression of IGF2BP2 and KLF7 in HNSCC was detected using clinical samples. To determine the biological role of IGF2BP2, we performed CCK-8, colony formation assay, Transwell migration assay, invasion assay, and orthotopic xenograft model experiments. Furthermore, we utilized a CRISPR/Cas9 gene-editing system, small-molecule inhibitors, ChIP-qPCR, and dual-luciferase reporter assays to investigate the molecular mechanisms of IGF2BP2 and its upstream transcription factors.ResultsOur study identified IGF2BP2 as a hub SE-associated gene that exhibited aberrant expression in HNSCC tissues. Increased expression of IGF2BP2 was observed to be linked with malignant progression and unfavorable prognosis in HNSCC patients. Both in vitro and in vivo experiments confirmed that IGF2BP2 promotes the tumorigenicity and metastasis of HNSCC by promoting cell proliferation, migration, and invasion. Mechanistically, the IGF2BP2-SE region displayed enrichment for H3K27Ac, BRD4, and MED1, which led to the inhibition of IGF2BP2 transcription and expression through deactivation of the SE-associated transcriptional program. Additionally, KLF7 was found to induce the transcription of IGF2BP2 and directly bind to its promoter and SE regions. Moreover, the abundance of KLF7 exhibited a positive correlation with the abundance of IGF2BP2 in HNSCC. Patients with high expression of both KLF7 and IGF2BP2 showed poorer prognosis. Lastly, we demonstrated that the small molecule inhibitor JQ1, targeting BRD4, attenuated the proliferation and metastatic abilities of HNSCC cells.ConclusionsOur study reveals the critical role of IGF2BP2 overexpression mediated by SE and KLF7 in promoting HNSCC progression. Targeting SE-associated transcriptional programs may represent a potential therapeutic strategy in managing HNSCC.

Identification of a prognostic model based on cuproptosis and ferroptosis-related genes in patients with head and neck squamous cell carcinoma

BackgroundHead and neck squamous cell carcinoma (HNSCC) is a common invasive malignant tumor that lacks powerful predictive or prognostic biomarkers. Ferroptosis and cuproptosis are two new forms of programmed cell death. Our study was aimed at constructing a prognostic model with a combination of cuproptosis and ferroptosis-related genes (CFRGs) for the early clinical detection of HNSCC. MethodsWe obtained the information of CFRGs, including the RNASeq data and corresponding clinical data in HNSCC patients from the TCGA and GEO databases. We assessed 28 CFRGs, and analyzed the relationship between those genes and their clinical features and prognosis of HNSCC. The consensus cluster analysis was employed to generate three CFRGclusters. Then, we investigated the association of molecular patterns and prognostic significance in these subtypes. The clinical indicators of the prognosis-related genes were identified and prognostic CFRG_score were constructed. We then built a predictive nomogram with confirmed consistency and reliability by calibration curve analysis. At last, we verified the expression of CFRGs in HNSCC tissues by qRT-PCR and immunohistochemical results. ResultsThe DEGs were different between the normal and HNSCC tumor tissues and we screened out 28 CFRGs related to the prognosis in HNSCC. Associations between the clinical information and prognosis were found in the molecular subtypes related to prognosis. We utilized enrichment analysis of the differential genes and showed that those DEGs were mostly enriched in the biological processes associated with the pathways of neurodegeneration-multiple diseases, Alzheimer disease, Prion disease, Parkinson disease and Amyotrophic lateral sclerosis. CFRG_score was established to predict the survival of HNSCC patients and found that higher CFRG_score suggested favorable OS for patients, indicating the prediction of better prognosis. Moreover, we created highly reliable nomogram which could predict well for the expected prognosis. In addition, we confirmed that the expression of EGFR, VEGFA, HSPA5, SLC3A2, CAV1 and CD44 were consistent with qRT-PCR and immunohistochemical analysis in HNSCC tissues by qRT-PCR. ConclusionsThis prognostic model based on prognostic differential CFRG_score is strongly related to clinical characteristics, prognosis, and therapy in HNSCC patients and could be used as a promising tool which is dedicated to guiding the treatment of HNSCC.

HSPA5, as a ferroptosis regulator, may serve as a potential therapeutic for head and neck squamous cell carcinoma

BackgroundHead and neck squamous cell carcinoma (HNSCC) is a ferroptosis sensitive tumor type with high mortality rate. However, it remains largely unknown whether ferroptosis influences the tumor cell in HNSCC. Materials and Methods: To investigate how ferroptosis regulators were differentially expressed between normal and tumor tissue, data related to HNSCC was downloaded from The Cancer Genome Atlas. The expression levels of key factors in HNSCC and the relationship between key factors and ferroptosis in HNSCC were conducted in vitro, and then analyzed to correlate with the differences in prognosis and survival. This was then combined with TNM staging data, and the migration effects of key factors in HNSCC were verified by scratch test and transwell test. Results: In this study, gene expression analysis and correlation studies between genes showed that HSPA5 was a potentially key associated ferroptosis regulator in HNSCC. Bioinformatics analysis showed that high expression of HSPA5 in HNSCC was positively correlated with poor prognosis and distal metastasis of HNSCC. In vitro immunohistochemistry and western blot tests confirmed that HSPA5 was highly expressed in HNSCC tissues and cell lines. In vitro inhibition of HSPA5 reduced the viability of HNSCC cells and increased ferroptosis. The results of scratch, transwell, and immunofluorescence tests showed that HSPA5 was related to the migration of HNSCC. In addition, a pan-cancer analysis showed that HSPA5 was also overexpressed in many types of cancer with poor prognoses. Conclusion: In total, our study demonstrates the critical role of ferroptosis regulators in HNSCC and that HSPA5, as a ferroptosis regulator, can be regarded as a key molecular target for designing new therapeutic regimens to control HNSCC metastasis and progression.

Abstract 4572: 3D-EXpress ex vivo platform using a biorepository of characterized fresh patient tumoroids allows development of rational combinations with drugs targeting DNA damage response and immune checkpoint blockade

Abstract Introduction: Nilogen Oncosystems’ 3D-EXpress platform allows for the detailed characterization offreshly resected patient tumor tissue for inclusion in studies investigating rational combinations of IOtherapeutics. Incorporating data on immune cell composition, tumor cell target expression, mutationalstatus, and HPV infection within tumoroids generated from HNSCC tissues provides tremendous input toidentify candidate tissues for therapeutic testing. The 3D-EXpress platform employed here providesdetailed observations investigating tumor cell viability using confocal microscopy and immune cellactivation indicated by increases in cytokine release. Materials and Methods: 3D tumoroids measuring 150 microns in size were generated from fresh patientHNSCC resection samples collected with proper patient consent and relevant IRB approval. Tumoroidswere never enzymatically dissociated, propagated, or reassembled to maintain the intact tumormicroenvironment (TME). The resulting tumoroids were cryopreserved in the Biorepository, where eachsample had associated patient demographic data, tumor grade, stage, mutational and HPV status inaddition to a detailed tumor immune profile and a FFPE representative sample in TMA format for targetselection. Pooled tumoroids for each patient tumor were treated ex vivo both singly with a selectiveWEE1 inhibitor, AZD1775 and an ATR blocker AZD6738 alone and in combination with a PD-1 immunecheckpoint inhibitor antibody nivolumab for 72h. Results and Summary: To quantify treatment-mediated tumor cell killing we used 3D high-contentconfocal imaging and a proprietary algorithm for data analysis. Additionally, culture supernatantsisolated from treated tumoroids were used for multiplex cytokine detection to monitor changes in theTME upon ex vivo treatment. Responses to treatments were further correlated with the composition ofinnate and adaptive TIL populations within each tumor as assessed by 21-color flow cytometry inaddition to tumor mutational status, HPV infection, smoking status, as well as EGFR and PD-L1expression levels analyzed by multiplex IF on associated TMA slides. Conclusion: These results demonstrate that targeting DNA damage response and immune checkpointblockade may provide a potential combination strategy for the treatment of HNSCC. Furthermore, weshowed that our 3D-EXpress ex vivo tumoroid model provides a unique platform to rapidly assess theefficacy of drugs and drug combinations in fresh patient tumor samples with intact TME. Correlation ofex vivo drug responses with characteristics of each tumor’s immune microenvironment and detailedclinicopathological data may allow to identify clinically relevant biomarkers to enable the most effectivetreatment strategies for individual patients in the clinic. Citation Format: Jared Ehrhart, Seth Currlin, Sharon Camacho, Samantha Hoffman, Angie Rivera, Soner Altiok. 3D-EXpress ex vivo platform using a biorepository of characterized fresh patient tumoroids allows development of rational combinations with drugs targeting DNA damage response and immune checkpoint blockade. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4572.

SEC11A contributes to tumour progression of head and neck squamous cell carcinoma

BackgroundHead and neck squamous cell carcinoma (HNSCC) is a prevalent disease that has a low survival rate and high recurrence risk. Our study aims to investigate the expression and role of SEC11A in HNSCC. MethodsThe expression of SEC11A was assessed in 18 pairs of cancerous and adjacent tissues by qRT-PCR and western blotting. Immunohistochemistry was performed in clinical specimen sections to evaluate the expression of SEC11A and its association with outcomes. Furthermore, the functional role of SEC11A in HNSCC tumor proliferation and progression was investigated using the in vitro cell model with lentivirus-mediated SEC11A knockdown. Colony formation and CCK8 assays were conducted to assess cell proliferation potential, while in vitro migration and invasion were examined using wound healing and transwell assays. To determine the tumor formation potential in vivo, a tumor xenograft assay was used. ResultsIn contrast to adjacent normal tissues, SEC11A expression was significantly elevated in HNSCC tissues. SEC11A was mainly localized in the cytoplasm, and its expression was significantly associated with patient prognosis. SEC11A was silenced using shRNA lentivirus in TU212 and TU686 cell lines, and the gene knockdown was confirmed. A series of functional assays demonstrated that SEC11A knockdown reduced cell proliferation, migration and invasion ability in vitro. In addition, the xenograft assay demonstrated that SEC11A knockdown significantly inhibited tumor growth in vivo. Tumor tissue sections of mice showed decreased proliferation potential in the shSEC11A xenografts cells by immunohistochemistry. ConclusionSEC11A knockdown decreased cell proliferation, migration and invasion in vitro and subcutaneous tumorigenesis in vivo. SEC11A is crucial to HNSCC proliferation and progression, and may serve as a new therapeutic target.

Development and validation of a GRGPI model for predicting the prognostic and treatment outcomes in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is among the most lethal and most prevalent malignant tumors. Glycolysis affects tumor growth, invasion, chemotherapy resistance, and the tumor microenvironment. Therefore, we aimed at identifying a glycolysis-related prognostic model for HNSCC and to analyze its relationship with tumor immune cell infiltrations. The mRNA and clinical data were obtained from The Cancer Genome Atlas (TCGA), while glycolysis-related genes were obtained from the Molecular Signature Database (MSigDB). Bioinformatics analysis included Univariate cox and least absolute shrinkage and selection operator (LASSO) analyses to select optimal prognosis-related genes for constructing glycolysis-related gene prognostic index(GRGPI), as well as a nomogram for overall survival (OS) evaluation. GRGPI was validated using the Gene Expression Omnibus (GEO) database. A predictive nomogram was established based on the stepwise multivariate regression model. The immune status of GRGPI-defined subgroups was analyzed, and high and low immune groups were characterized. Prognostic effects of immune checkpoint inhibitor (ICI) treatment and chemotherapy were investigated by Tumor Immune Dysfunction and Exclusion (TIDE) scores and half inhibitory concentration (IC50) value. Reverse transcription-quantitative PCR (RT-qPCR) was utilized to validate the model by analyzing the mRNA expression levels of the prognostic glycolysis-related genes in HNSCC tissues and adjacent non-tumorous tissues. Five glycolysis-related genes were used to construct GRGPI. The GRGPI and the nomogram model exhibited robust validity in prognostic prediction. Clinical correlation analysis revealed positive correlations between the risk score used to construct the GRGPI model and the clinical stage. Immune checkpoint analysis revealed that the risk model was associated with immune checkpoint-related biomarkers. Immune microenvironment and immune status analysis exhibited a strong correlation between risk score and infiltrating immune cells. Gene set enrichment analysis (GSEA) pathway enrichment analysis showed typical immune pathways. Furthermore, the GRGPIdel showed excellent predictive performance in ICI treatment and drug sensitivity analysis. RT-qPCR showed that compared with adjacent non-tumorous tissues, the expressions of five genes were significantly up-regulated in HNSCC tissues. The model we constructed can not only be used as an important indicator for predicting the prognosis of patients but also had an important guiding role for clinical treatment.

Cuproptosis-related LncRNAs are potential prognostic and immune response markers for patients with HNSCC via the integration of bioinformatics analysis and experimental validation.

Head and neck squamous cell carcinoma (HNSCC) is a malignant neoplasm typically induced by alcohol and tobacco consumption, ranked the sixth most prevalent cancer globally. This study aimed to establish a cuproptosis-related lncRNA predictive model to assess the clinical significance in HNSCC patients. The Cancer Genome Atlas (TCGA) database was utilized to download cuproptosis-related genes, lncRNAs profiles, and selected clinical information of 482 HNSCC samples. Cuproptosis-related lncRNAs were analyzed by Pearson correlation method, with the least absolute shrinkage and selection operator (LASSO) and univariate/multivariate Cox analyses performed to establish the cuproptosis-related lncRNA predictive model. Subsequently, the time-dependent receiver operating characteristics (ROC) and Kaplan-Meier analysis were applied to assess its prediction ability, and the model was verified by a nomogram, univariate/multivariate Cox analysis, and calibration curves. Furthermore, the principal component analysis (PCA), immune analysis, and gene set enrichment analyses (GSEA) were performed, and the 50% inhibitory concentration (IC50) prediction in the risk groups was calculated. Furthermore, the expression of six cuproptosis-related lncRNAs in HNSCC and paracancerous tissues was detected by quantitative real-time PCR (qRT-PCR). A total of 467 lncRNAs were screened as cuproptosis-associated lncRNAs in HNSCC tissues to establish an eight cuproptosis-related lncRNA prognostic signature consisting of AC024075.3, AC090587.2, AC116914.2, AL450384.2, CDKN2A-DT, FAM27E3, JPX, and LNC01089. For the high-risk group, the results demonstrated a satisfactory predicting performance with considerably worse overall survival (OS). Multivariate Cox regression confirmed that the risk score was a reliable predictive factor (95% CI: 1.089-1.208, hazard ratio =1.147), with the area of 1-, 3-, and 5-year OS under the ROC curve of 0.690, 0.78524, and 0.665, respectively. The differential analysis revealed that JPX was significantly upregulated in HNSCC tissues, while AC024075.3, AC090587.2, AC116914.2, AL450384.2, CDKN2A-DT were downregulated in HNSCC tissues by qRT-PCR assays. In addition, this gene signature was also associated with some immune-related pathways and immune cell infiltration and affected the anti-cancer immune response. Furthermore, Bexarotene, Bleomycin, Gemcitabine, etc., were identified as potential therapeutic compounds for HNSCC. This novel cuproptosis-related lncRNAs prognostic signature could predict prognosis and help propose novel individual therapeutic targets for HNSCC.

An ion-channel-gene-based prediction model for head and neck squamous cell carcinoma: Prognostic assessment and treatment guidance.

Head and neck squamous cell carcinoma (HNSCC) is a very diverse malignancy with a poor prognosis. The purpose of this study was to develop a new signature based on 12 ion channel genes to predict the outcome and immune status of HNSCC patients. Clinicopathological information and gene sequencing data of HNSCC patients were generated from the Cancer Genome Atlas and Gene Expression Omnibus databases. A set of 323 ion channel genes was obtained from the HUGO Gene Nomenclature Committee database and literature review. Using univariate Cox regression analysis, the ion channel genes related to HNSCC prognosis were identified. A prognostic signature and nomogram were then created using machine learning methods. Kaplan-Meier analysis was used to explore the relevance of the risk scores and overall survival (OS). We also investigated the association between risk scores, tumor immune infiltration, and gene mutational status. Finally, we detected the expression levels of the signature genes by quantitative real-time polymerase chain reaction, western blotting, and immunohistochemistry. We separated the patients into high- and low-risk groups according to the risk scores computed based on these 12 ion channel genes, and the OS of the low-risk group was significantly longer (p<0.001). The area under the curve for predicting 3-year survival was 0.729. Univariate and multivariate analyses showed that the 12-ion-channel-gene risk model was an independent prognostic factor. We also developed a nomogram model based on risk scores and clinicopathological variables to forecast outcomes. Furthermore, immune cell infiltration, gene mutation status, immunotherapy response, and chemotherapeutic treatment sensitivity were all linked to risk scores. Moreover, high expression levels of ANO1, AQP9, and BEST2 were detected in HNSCC tissues, whereas AQP5, SCNN1G, and SCN4A expression was low in HNSCC tissues, as determined by experiments. The 12-ion-channel-gene prognostic signatures have been demonstrated to be highly efficient in predicting the prognosis, immune microenvironment, gene mutation status, immunotherapy response, and chemotherapeutic sensitivity of HNSCC patients.

Circ_0000045 promotes proliferation, migration, and invasion of head and neck squamous cell carcinomas via regulating HSP70 and MAPK pathway

ObjectiveHead and neck squamous cell carcinoma (HNSCC) is one severe malignancy driven by complex cellular and signaling mechanisms. However, the roles of circular RNAs (circRNAs) in HNSCC’s development remains poorly understood. Therefore, this study investigated the functions of differentially expressed circRNAs in regulating HNSCC cell functions.MethodsDifferentially expressed circRNAs were characterized through RNA sequencing in HNSCC tissues. CircRNA’s identity was then confirmed using RT-PCR and Sanger’s sequencing. Next, expression levels of circRNA and mRNA were detected by qRT-PCR, after which protein abundances were measured by Western blotting. Subsequently, the proliferation, migration, and invasion of HNSCC cells was assessed by MTS, wound healing, and Transwell system, respectively, followed by identification of circRNA-binding proteins in HNSCC cells by circRNA pull-down, coupled with mass spectrometry.ResultsGreat alterations in circRNA profiles were detected in HNSCC tissues, including the elevated expression of circ_0000045. As observed, silencing of circ_0000045 effectively repressed the proliferation, migration, and invasion of HNSCC cell lines (FaDu and SCC-9). Contrarily, circ_0000045’s overexpression promoted the proliferation, migration, and invasion in FaDu and SCC-9 cells. Results also showed that circ_0000045 was associated with multiple RNA-binding proteins in HNSCC cells, such as HSP70. Moreover, circ_0000045 knockdown enhanced HSP70 expression and inhibited JNK2 and P38’s expression in HNSCC cells, which were oppositely regulated by circ_0000045’s overexpression.ConclusionThe high expression of circ_0000045; therefore, promoted cell proliferation, migration, and invasion during HNSCC’s development through regulating HSP70 protein and mitogen-activated protein kinase signaling.

Growth regulated oncogene-α contribute to EMT/MMPs pathway by binding its receptors in head and neck squamous cell carcinoma

Squamous cell carcinoma (SCC) is the most common malignant tumor of the head and neck and generally detected in the late stages when the cancer has advanced, and therefore has a poor prognosis and survival rate. A high expression of growth-related oncogene alpha (Groα) is associated with tumor metastasis and invasion and the poor survival rate of patients. Microarray reveals that Groα exhibits a cancer-specific response in HNSCC. Quantitative real-time PCR (qRT-PCR) results concerning the mRNA expression of Groα in HNSCC tissues; indicate that Groα was more highly expressed in HNSCC than in non-cancerous matched tissue (NCMT). The serum of HNSCC patients and healthy subjects demonstrates that the expression of Groα in the HNSCC patients significantly exceeded than in healthy subjects. Furthermore, exposure Groα to stimulated the proliferation, clonogenicity and migration with HNSCC cells (SCC4, SCC9, SCC25 and OECM-1), yielding a stronger response than in non-malignant HaCaT and DOK cells. A high expression of Groα and its receptors CXCR1/2 (chemokine (C-X-C motif) receptor) in HNSCC tissues are highly correlated with tumor progression stage and metastasis. Following the treatment of SCC25 and OECM-1 cells with Groα, β-catenin, matrix metalloproteinases (MMP)-2, MMP-7 and MMP-9 expressions significantly increased but E-cadherin expression was slightly decreased, suggesting that the EMT and metastasis processes were activated by Groα. These findings constitute the first evidence that Groα promotes epithelial mesenchymal transition (EMT) and MMPs expressions in HNSCC via activating CXCR1/2, suggesting a role for Groα in mediating metastasis and its potential as a therapeutic target.

A Robust Metabolic Enzyme-Based Prognostic Signature for Head and Neck Squamous Cell Carcinoma.

BackgroundHead and neck squamous cell carcinoma (HNSCC) is still a menace to public wellbeing globally. However, the underlying molecular events influencing the carcinogenesis and prognosis of HNSCC are poorly known.MethodsGene expression profiles of The Cancer Genome Atlas (TCGA) HNSCC dataset and GSE37991 were downloaded from the TCGA database and gene expression omnibus, respectively. The common differentially expressed metabolic enzymes (DEMEs) between HNSCC tissues and normal controls were screened out. Then a DEME-based molecular signature and a clinically practical nomogram model were constructed and validated.ResultsA total of 23 commonly upregulated and 9 commonly downregulated DEMEs were identified in TCGA HNSCC and GSE37991. Gene ontology analyses of the common DEMEs revealed that alpha-amino acid metabolic process, glycosyl compound metabolic process, and cellular amino acid metabolic process were enriched. Based on the TCGA HNSCC cohort, we have built up a robust DEME-based prognostic signature including HPRT1, PLOD2, ASNS, TXNRD1, CYP27B1, and FUT6 for predicting the clinical outcome of HNSCC. Furthermore, this prognosis signature was successfully validated in another independent cohort GSE65858. Moreover, a potent prognostic signature-based nomogram model was constructed to provide personalized therapeutic guidance for treating HNSCC. In vitro experiment revealed that the knockdown of TXNRD1 suppressed malignant activities of HNSCC cells.ConclusionOur study has successfully developed a robust DEME-based signature for predicting the prognosis of HNSCC. Moreover, the nomogram model might provide useful guidance for the precision treatment of HNSCC.

Discrepancy in p16 expression in patients with HPV-associated head and neck squamous cell carcinoma in Thailand: clinical characteristics and survival outcomes

BackgroundLower prevalence HPV infection has been previously reported in Thai population when compared with Western countries. p16 expression indicates HPV-associated oropharyngeal squamous cell carcinoma (OPSCC), but not non-OPSCC. We therefore evaluated the characteristic and association of p16 and HPV in Thai patients with HNSCC.MethodsWe used immunohistochemistry and qPCR, respectively, to detect p16 and HPV DNA in archrival formalin-fixed paraffin-embedded HNSCC tissues. Patient characteristics and survival were analyzed.Resultsp16 expression was detected in tumors of 72 of 662 (10.9%) patients with HNSCC and was significantly associated with higher-grade histology, advanced nodal stage, and oropharynx. p16 was expressed in 28 and 6.5% of patients with OPSCC or non-OPSCC, respectively, and HPV DNA was detected in 15.6 and 1% of patients, respectively. Using p16 as a surrogate for HPV status, sensitivities were 80 and 25% in OPSCC and non-OPSCC, respectively. Positive and negative predictive rates of OPSCC were 38 and 95%. Discordance rates between HPV and p16 were 23 and 7% in OPSCC and non-OPSCC, respectively. Overall survival (OS) were significantly longer in both p16-positive OPSCC (p = 0.049), and non-OPSCC (p = 0.003).ConclusionsLow prevalence of p16 and HPV associated OPSCC and non-OPSCC were confirmed in Thai patients. High discordance and low positive predictive rates of p16 were observed in HPV-associated OPSCC. p16 was a significant prognostic factor for OS for patients with OPSCC or non-OPSCC. Therefore, HPV testing should be performed to assess the association of HPV with HNSCC regardless of p16 expression.

Development and validation of a RNA binding protein-associated prognostic model for head and neck squamous cell carcinoma.

Evidence shows that defects in RNA-binding proteins (RBPs) are closely related to the occurrence and development of HNSCC. We obtained 502 tumors and 44 normal samples from the TCGA database, among which 190 differentially expressed RBPs were screened. Finally, a prognostic model containing nine RBPs (CELF2, CPEB1, DDX39B, EIF3L, EZH2, KHDRBS3, RNASE10, RNASE3 and SIDT1) was produced. Further analysis showed that the overall survival rate in the high-risk group was lower than that in the low-risk group. The area under the ROC curve (AUC) in the training and testing groups was significant (3-year AUC, 0.735 vs 0.796; 5-year AUC, 0.821 vs 0.804). In addition, a comprehensive analysis of nine identified RBPs showed that most of them were related to the OS of HNSCC patients, and three of them (CELF2, EZH2, and SIDT1) were differentially expressed in HNSCC and control tissues at the protein level. In addition, our data revealed that the identified RBPs are highly interconnected, with high frequency copy number changes in HNSCC samples. GSEA indicated that the abnormal biological processes related to RNA and the activation of some classical tumor signaling pathways were important driving forces for the development of HNSCC. Our results provide novel insights into the pathogenesis of HNSCC, among which nine RBP markers have potential application value in clinical decision-making and individualized treatment of HNSCC.