TCGA Head And Neck Squamous Cell Carcinoma Research Articles

Circulating tumour DNA detects somatic variants contributing to spatial and temporal intra-tumoural heterogeneity in head and neck squamous cell carcinoma

BackgroundAs circulating tumour DNA (ctDNA) liquid biopsy analysis is increasingly incorporated into modern oncological practice, establishing the impact of genomic intra-tumoural heterogeneity (ITH) upon data output is paramount. Despite advances in other cancer types the evidence base in head and neck squamous cell carcinoma (HNSCC) remains poor. We sought to investigate the utility of ctDNA to detect ITH in HNSCC.MethodsIn a pilot cohort of 9 treatment-naïve HNSCC patients, DNA from two intra-tumoural sites (core and margin) was whole-exome sequenced. A 9-gene panel was designed to perform targeted sequencing on pre-treatment plasma cell-free DNA and selected post-treatment samples.ResultsRates of genomic ITH among the 9 patients was high. COSMIC variants from 19 TCGA HNSCC genes demonstrated an 86.9% heterogeneity rate (present in one tumour sub-site only). Across all patients, cell-free DNA (ctDNA) identified 12.9% (range 7.5-19.8%) of tumour-specific variants, of which 55.6% were specific to a single tumour sub-site only. CtDNA identified 79.0% (range: 55.6-90.9%) of high-frequency variants (tumour VAF>5%). Analysis of ctDNA in serial post-treatment blood samples in patients who suffered recurrence demonstrated dynamic changes in both tumour-specific and acquired variants that predicted recurrence ahead of clinical detection.ConclusionWe demonstrate that a ctDNA liquid biopsy identified spatial genomic ITH in HNSCC and reliably detected high-frequency driver mutations. Serial sampling allowed post-treatment surveillance and early identification of treatment failure.

Single cell analysis unveils B cell-dominated immune subtypes in HNSCC for enhanced prognostic and therapeutic stratification

Head and neck squamous cell carcinoma (HNSCC) is characterized by high recurrence or distant metastases rate and the prognosis is challenging. There is mounting evidence that tumor-infiltrating B cells (TIL-Bs) have a crucial, synergistic role in tumor control. However, little is known about the role TIL-Bs play in immune microenvironment and the way TIL-Bs affect the outcome of immune checkpoint blockade. Using single-cell RNA sequencing (scRNA-seq) data from the Gene Expression Omnibus (GEO) database, the study identified distinct gene expression patterns in TIL-Bs. HNSCC samples were categorized into TIL-Bs inhibition and TIL-Bs activation groups using unsupervised clustering. This classification was further validated with TCGA HNSCC data, correlating with patient prognosis, immune cell infiltration, and response to immunotherapy. We found that the B cells activation group exhibited a better prognosis, higher immune cell infiltration, and distinct immune checkpoint levels, including elevated PD-L1. A prognostic model was also developed and validated, highlighting four genes as potential biomarkers for predicting survival outcomes in HNSCC patients. Overall, this study provides a foundational approach for B cells-based tumor classification in HNSCC, offering insights into targeted treatment and immunotherapy strategies.

Abstract 3061: A positive feedback loop between GLS1 and c-Myc drives tumor aggressiveness

Abstract Both GLS1 and c-Myc are upregulated in head and neck squamous cell carcinoma (HNSCC) primary tumors and further increased in metastatic tumors. However, the regulation between these two molecules remains largely unknown. GLS1 is a critical enzyme that regulates glutamate, which plays an important role in cancer metabolism that supports cancer growth and survival. Our bioinformatic analysis of the TCGA HNSCC cohort revealed a strong correlation between c-Myc and GLS1 expression. We describe the importance of c-Myc in regulating GLS1 and vice versa. c-Myc protein directly binds to the promoter of the GLS1 gene and upregulates its expression at the transcriptional level. Interestingly, blocking GLS1 signaling in HNSCC cells by lentiviral shRNA knockdown or CB-839 treatment downregulates USP1, one of the best characterized human DUBs, which in turn reduces c-Myc protein stability via the ubiquitin-proteasome pathway. The GLS1-c-Myc axis thus represents a novel positive feedback loop that is critical for driving the aggressiveness of HNSCC. As the treatment of HNSCC remains a major challenge, these novel findings provide the molecular basis for combining GLS1-specific inhibitors with c-Myc-targeted therapy for the treatment of HNSCC patients. Citation Format: Jianqiang Yang, Fanghui Chen, Fan Yang, Yong Teng. A positive feedback loop between GLS1 and c-Myc drives tumor aggressiveness [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 3061.

Combined IL6 and CCR2 blockade potentiates antitumor activity of NK cells in HPV-negative head and neck cancer

BackgroundWhile T cell-activating immunotherapies against recurrent head and neck squamous cell carcinoma (HNSCC) have shown impressive results in clinical trials, they are often ineffective in the majority of patients. NK cells are potential targets for immunotherapeutic intervention; however, the setback in monalizumab-based therapy in HNSCC highlights the need for an alternative treatment to enhance their antitumor activity.MethodsSingle-cell RNA sequencing (scRNA-seq) and TCGA HNSCC datasets were used to identify key molecular alterations in NK cells. Representative HPV-positive ( +) and HPV-negative ( −) HNSCC cell lines and orthotopic mouse models were used to validate the bioinformatic findings. Changes in immune cells were examined by flow cytometry and immunofluorescence.ResultsThrough integration of scRNA-seq data with TCGA data, we found that the impact of IL6/IL6R and CCL2/CCR2 signaling pathways on evasion of immune attack by NK cells is more pronounced in the HPV − HNSCC cohort compared to the HPV + HNSCC cohort. In orthotopic mouse models, blocking IL6 with a neutralizing antibody suppressed HPV − but not HPV + tumors, which was accompanied by increased tumor infiltration and proliferation of CD161+ NK cells. Notably, combining the CCR2 chemokine receptor antagonist RS504393 with IL6 blockade resulted in a more pronounced antitumor effect that was associated with more activated intratumoral NK cells in HPV − HNSCC compared to either agent alone.ConclusionsThese findings demonstrate that dual blockade of IL6 and CCR2 pathways effectively enhances the antitumor activity of NK cells in HPV-negative HNSCC, providing a novel strategy for treating this type of cancer.

TSPO is a potential independent prognostic factor associated with cellular respiration and p16 in head and neck squamous cell carcinoma.

Treatment resistance and relapse are common problems in head and neck squamous cell carcinoma (HNSCC). Except for p16, no clinically accepted prognostic biomarkers are available for HNSCC. New biomarkers predictive of recurrence and survival are crucial for optimal treatment planning and patient outcome. High translocator protein (TSPO) levels have been associated with poor survival in cancer, but the role of TSPO has not been extensively evaluated in HNSCC. TSPO expression was determined in a large population-based tissue microarray cohort including 611 patients with HNSCC and evaluated for survival in several clinicopathological subgroups. A TCGA HNSCC cohort was used to further analyze the role of TSPO in HNSCC. TSPO expression was downregulated in more aggressive tumors. Low TSPO expression associated with worse 5-year survival and was an independent prognostic factor for disease-specific survival. Subgroup analyses showed that low TSPO expression associated with worse survival particularly in p16-positive oropharyngeal cancer. In silico analyses supported the prognostic role of TSPO. Cellular respiration had the highest significance in pathway analyses for genes expressed positively with TSPO. Decreased TSPO expression associates with poor prognosis in HNSCC. TSPO is a prognostic biomarker in HNSCC to potentially guide treatment stratification especially in p16-positive oropharyngeal cancer.

Identification and validation of a novel signature based on T cell marker genes to predict prognosis, immunotherapy response and chemotherapy sensitivity in head and neck squamous carcinoma by integrated analysis of single-cell and bulk RNA-sequencing

T cells are among the most potent anti-tumor cells that are found in humans. Our study sought to develop a reliable signature incorporating T cell marker genes (TMGs) for predicting the prognosis and therapy responsiveness of head and neck squamous cell carcinoma (HNSCC) patients. We downloaded scRNA-seq data from the GSE181919 to identify TMGs. Subsequently, we devised a 12 TMG signature in the TCGA HNSCC cohort by using LASSO analysis. Patients with high-risk scores were shown to experience unfavorable progression-free survival, disease-specific survival, and overall survival, which was validated in the GSE65858 cohort. Additionally, the nomogram integrated risk score and clinical features are more suitable for clinical application. The enrichment analyses of both pathways and functions showed that high- and low-risk patients had functionally related distinctions. Furthermore, analysis of the immunological landscape confirmed that the low-risk patients had a larger percentage of infiltrating immune cells as well as a higher incidence rate of immune-related events. In the meantime, a greater IPS score and expression of immune checkpoint genes suggested significantly favorable responsiveness to immunotherapy in low-risk patients. On the other hand, the high-risk patients had a greater degree of sensitivity to the chemotherapy agents, which included paclitaxel, gemcitabine, docetaxel, and cisplatin. Our finding revealed that this TMG signature independently functioned as a prognostic marker and guided individualized immunotherapy and chemotherapy selection for patients with HNSCC.

Machine learning driven index of tumor multinucleation correlates with survival and suppressed anti-tumor immunity in head and neck squamous cell carcinoma patients

ObjectivesMatching treatment intensity to tumor biology is critical to precision oncology for head and neck squamous cell carcinoma (HNSCC) patients. We sought to identify biological features of tumor cell multinucleation, previously shown by us to correlate with survival in oropharyngeal (OP) SCC using a machine learning approach. Materials and methodsHematoxylin and eosin images from an institutional OPSCC cohort formed the training set (DTr). TCGA HNSCC patients (oral cavity, oropharynx and larynx/hypopharynx) formed the validation set (DV). Deep learning models were trained in DTr to calculate a multinucleation index (MuNI) score. Gene set enrichment analysis (GSEA) was then used to explore correlations between MuNI and tumor biology. ResultsMuNI correlated with overall survival. A multivariable nomogram that included MuNI, age, race, sex, T/N stage, and smoking status yielded a C-index of 0.65, and MuNI was prognostic of overall survival (2.25, 1.07–4.71, 0.03), independent of the other variables. High MuNI scores correlated with depletion of effector immunocyte subsets across all HNSCC sites independent of HPV and TP53 mutational status although the correlations were strongest in wild-type TP53 tumors potentially due to aberrant mitotic events and activation of DNA-repair mechanisms. ConclusionMuNI is associated with survival in HNSCC across subsites. This may be driven by an association between high levels of multinucleation and a suppressive (potentially exhausted) tumor immune microenvironment. Mechanistic studies examining the link between multinucleation and tumor immunity will be required to characterize biological drivers of multinucleation and their impact on treatment response and outcomes.

Prognostic biomarker IL17A correlated with immune infiltrates in head and neck cancer

BackgroundThe underlying functions and mechanisms of the Th17 pathway in Head and neck squamous cell carcinoma (HNSCC) progression and tumor immunology are still unclear. We investigated the correlation between IL17A expression and certain clinical parameters, tumor-infiltrating immune cells (TIICs) in TCGA HNSCC samples.MethodsHNSCC files from the TCGA database were analyzed to obtain data on immune system infiltrates, gene expression, and related clinical information. R (Version 3.6.3) software, GEPIA, and TIMER online analysis tools were used to profile the relationship between the expression of IL17A and the prognosis, clinical stages, survival status and immune cell tumor-infiltrating levels of HNSCC patients. GEPIA and TIMER online analysis tools were used to verify the data.ResultsThe expression of IL17A was significantly decreased in tumor tissues from HNSCC. IL17A expression was associated with M, N stage, lymphovascular invasion, and patients OS event. GSEA revealed that IL17A was closely related to humoral immune response, T cells response, and cytokine signal. TCGA database and TIMER online analysis indicated that the B cells and T cells levels were correlated with IL17A. The correlation between IL17A expression and correlated genes was analyzed.ConclusionsIL-17A plays a key role in HNSCC. The levels of IL17A are important values for the determination of the occurrence and development of the HNSCC. The IL17A and correlated genes may be potential immunotherapeutic targets for HNSCC.

Transcriptome analysis reveals the prognostic and immune infiltration characteristics of glycolysis and hypoxia in head and neck squamous cell carcinoma

BackgroundThis study aims to construct a new prognostic gene signature in survival prediction and risk stratification for patients with Head and neck squamous cell carcinoma (HNSCC).MethodThe transcriptome profiling data and hallmark gene sets in the Molecular Signatures Database was used to explore the cancer hallmarks most relevant to the prognosis of HNSCC patients. Differential gene expression analysis, weighted gene co-expression network analysis, univariate COX regression analysis, random forest algorithm and multiple combinatorial screening were used to construct the prognostic gene signature. The predictive ability of gene signature was verified in the TCGA HNSCC cohort as the training set and the GEO HNSCC cohorts (GSE41613 and GSE42743) as the validation sets, respectively. Moreover, the correlations between risk scores and immune infiltration patterns, as well as risk scores and genomic changes were explored.ResultsA total of 3391 differentially expressed genes in HNSCC were screened. Glycolysis and hypoxia were screened as the main risk factors for OS in HNSCC. Using univariate Cox analysis, 97 prognostic candidates were identified (P < 0.05). Top 10 important genes were then screened out by random forest. Using multiple combinatorial screening, a combination with less genes and more significant P value was used to construct the prognostic gene signature (RNF144A, STC1, P4HA1, FMNL3, ANO1, BASP1, MME, PLEKHG2 and DKK1). Kaplan–Meier analysis showed that patients with higher risk scores had worse overall survival (p < 0.001). The ROC curve showed that the risk score had a good predictive efficiency (AUC > 0.66). Subsequently, the predictive ability of the risk score was verified in the validation sets. Moreover, the two-factor survival analysis combining the cancer hallmarks and risk scores suggested that HNSCC patients with the high hypoxia or glycolysis & high risk-score showed the worst prognosis. Besides, a nomogram based on the nine-gene signature was established for clinical practice. Furthermore, the risk score was significantly related to tumor immune infiltration profiles and genome changes.ConclusionThis nine-gene signature associated with glycolysis and hypoxia can not only be used for prognosis prediction and risk stratification, but also may be a potential therapeutic target for patients with HNSCC.

An Epithelial-Mesenchymal Transition Hallmark Gene-Based Risk Score System in Head and Neck Squamous-Cell Carcinoma.

BackgroundEpithelial-to-mesenchymal transition (EMT) program plays a critical role in cancer. Thus, we attempted to generate a risk score system according to the expression pattern of different EMT hallmark genes in head and neck squamous-cell carcinoma (HNSC).MethodsDifferentially expressed EMT hallmark genes were screened to generate a risk score (RS) on TCGA HNSC dataset. The relative prognostic value of the RS compared to clinicopathological characteristics was explored using multivariable Cox analysis. Functional enrichment analysis was performed to reveal the biological characteristics. An external dataset was applied to validate the prognostic value of the RS.ResultsNine genes constituted the EMT hallmark gene-based RS, which is significantly associated with poor prognosis and could successfully divide patients with HNSC into high- and low-risk groups. The RS was also an independent prognostic indicator compared to routine clinical factors.ConclusionWe proposed and validated a nine-EMT hallmark gene-based risk score system in HNSC.

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.

Comprehensive analysis of radiosensitivity in head and neck squamous cell carcinoma

BackgroundRadioresistance is a major barrier to the successful treatment of head and neck squamous cell carcinoma (HNSCC). MethodsWe took advantage of different types of data, including single-cell sequencing data, bulk tissue sequencing data and deconvolution data, to conduct a comprehensive analysis of HNSCC radiosensitivity at the cellular, patient, and cell type levels. Single-cell transcriptomes for 1388 primary cancer cells from a previous study were analysed. The TCGA HNSCC dataset including 499 primary HNSCC samples with RNA-seq data, DNA methylation data and clinical information were used for bulk tissue sequencing analyses and deconvolution. ResultsWe found that radiosensitivity clustering of HNSCC cells was highly consistent with molecular typing, where cancer cells of the atypical subtype exhibited a higher sensitivity than those of the classical and basal subtypes. The common radioresistant gene modules of the classical and basal subtypes were mainly associated with cell division and cell cycle regulation; the classical subtype specific radioresistant module was mainly associated with metabolic pathways; and the basal radioresistant subtype specific modules included two epithelial differentiation-related modules and a module mainly associated with endoplasmic reticulum, apoptosis and focal adhesion. We developed a radioresistance score using genes that affect both the cancer cell response to radiation and the patient response to radiotherapy. An enhanced cancer-immune interaction through the PD1-PDL1/PDL2 and TIM3-Galectin9 pathways was observed in radioresistant tumours, with foldchange = 2.88 (PD1), 1.44 (PDL1), 3.22 (PDL2), 1.47 (TIM3), 1.88 (Galectin9) respectively and FDR < 0.001. Transcriptional activities related to the hypoxia response, p53 pathway, NF-kappa-B pathway and inflammatory response were abnormally activated in the radioresistant tumours (FDR < 0.05). ConclusionsThis study comprehensively discussed the radioresistance of HNSCC, identified a group of HNSCCs that were likely to benefit from combined radiotherapy and immune checkpoint blockade, and proposed new targets for the treatment of radioresistant HNSCC.

Prognostic Value of a Ten-Gene Signature in HNSCC Patients Based on Tumor-Associated Macrophages Expression Profiling.

Tumor-associated macrophages (TAMs) are regarded as the most abundantly infiltrating immune cells around the tumor microenvironment (TME) in head and neck squamous cell carcinoma (HNSCC), which plays an essential role in immunosuppression and tumorigenesis. In the TCGA HNSCC cohort, 500 patients with clinical-pathological information and RNA sequence expression were randomly assigned to training for lasso regression and validation for verification, respectively. A TAM-based ten-gene signature (TBGs) was constructed, which divided the patients into high-risk and low-risk groups, could predict overall survival (OS) of HNSCC patients in the training dataset (p = 3.527e–05) and validation dataset (p = 3.785e–02). The result of Cox univariate and multivariate regression analyses showed that the risk score of TBGs could be an independent prognostic factor in HNSCC. ROC curve confirmed that the risk score of TBGs has good sensitivity and specificity for prognosis prediction (AUC = 0.659) and was also verified by the validation dataset (AUC = 0.621). We obtained key risk transcription factors (TFs)—EHF and SNAI2—by correlation analysis with TBGs. Moreover, we ran a gene set enrichment analysis (GSEA) to speculate that TBGs act on interstitial remodeling, tumor killing, metabolic reprogramming, and tumor immune-related pathways. Finally, we combined clinical–pathological features and risk score of TBGs to establish clinical nomograms, and calibration curves verified the accuracy of long-term clinical prognosis in the two datasets (C-index of 5-year OS = 0.721 and 0.716). In general, the TBGs we obtained may accurately predict the prognosis of HNSCC patients to provide personalized treatment.

Abstract 1792: MAPK1 p.D321N mutation confers sensitive to erlotinib in head and neck squamous cell carcinoma (HNSCC)

Abstract The annual incidence of head and neck squamous cell carcinoma (HNSCC) is ~0.88 million worldwide (IARC, WHO 2018). Despite great success of precision medicine in major cancers such as lung cancer and breast cancers, there are limited precision therapies for HNSCC. We previously reported an HNSCC patient whose tumor harbored a somatic MAPK1 p.E322K mutation demonstrating exceptional clinical response to erlotinib, an EGFR inhibitor. Subsequent laboratory investigation confirmed the ability of MAPK1 p.E322K to hyperactivate EGFR, thus causing erlotinib sensitivity in HNSCC models, both in vitro and in vivo. Here, we sequenced 105 tumors from Hong Kong (HK) HNSCC patients by next-generation sequencing (NGS) on MAPK1 gene and we found a 5.71% rate (6/105 tumors) of MAPK1 somatic mutation in HK-HNSCC. This rate appears to be relatively higher than the 1.75% (9/521 tumors) rate in the US-TCGA HNSCC cohort (P=0.0288). Apart from the MAPK1 p.E322K mutation found in the US-TCGA HNSCC cohort, we identified novel MAPK1 p.R135K and p.D321N mutations in our HK cohort which were absent in the US TCGA HNSCC. Pan-cancer mapping of MAPK1 somatic mutations revealed that MAPK1 p.R135K and p.D321N mutations are also recurrent hotspot mutation in TCGA pan-cancers (32 cancer types). Strikingly, based on the X-ray crystallography of the human MAPK1 protein, R135 and D321 amino acid residues both reside on the same kinase interaction motif (KIM) domain as E322 (all are within a 13Å distance in 3D). Thus, we further investigated if MAPK1 p.R135K and p.D321N mutations might function similarly as MAPK1 p.E322K mutant in terms of their abilities to drive HNSCC growth, and erlotinib sensitivity in HNSCC models. We employed the same FaDu retroviral infection method to study these mutations as we did previously, and we found that MAPK1 p.D321N (P&amp;lt;0.0001) was a driver for HNSCC proliferation, while MAPK1 p.R135K was not (all vs. MAPK1-wildtype). Subsequent in vivo erlotinib treatment experiments with xenografts bearing these mutants and MAPK1-wildtype showed that MAPK1 p.D321N tumors demonstrated heightened sensitivity to erlotinib with marked reduction of tumor size, tumor cell positivity, as well as membranous pEGFR expression when compared to vehicle treatment (P=0.0037). Whereas MAPK1 p.R135K tumors only showed a trend for erlotinib sensitivity (P=0.0646), with reduction in membranous p-EGFR expression by erlotinib in the tumors. Thus, MAPK1 p.D321N mutation confers erlotinib-sensitivity in HNSCC system. Further clinical investigation is warranted to further explore the relationship between various MAPK1 mutations and erlotinib sensitivity in HNSCC patients. Citation Format: Hoi Lam Ngan, Peony Hiu Yan Poon, Yu-Xiong Su, Jason Yik Kuen Chan, Kwok-Wai Lo, Chun Kit Yeung, Yuchen Liu, Eileen Wong, Hui Li, Chin Wang Lau, Wenying Piao, Vivian Wai Yan Lui. MAPK1 p.D321N mutation confers sensitive to erlotinib in head and neck squamous cell carcinoma (HNSCC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1792.

Abstract 2442: Identification of a novel role for the β-catenin/CBP signaling in epigenetic regulation of the N-glycosylation gene, DPAGT1, in head and neck cancer

Abstract Aberrant activation of the DPAGT1 gene, encoding an essential enzyme in the metabolic pathway of protein N-glycosylation, has been shown to be associated with head and neck squamous cell carcinoma (HNSCC). We have shown that DPAGT1 inhibits intercellular adhesion and functions in a positive feedback loop with Wnt/β-catenin signaling, and that the nuclear β-catenin/CBP signaling underlies the progression of HNSCC to advanced disease. However, the tumor promoting effects of DPAGT1 and its molecular links to the nuclear β-catenin/CBP axis are not well defined. We carried out genomic and functional analyses of DPAGT1 perturbation in indolent (CAL27) and metastatic (HSC-3) HNSCC cells, and in orthotopic HSC-3-derived xenografts in mice. We further generated and annotated DPAGT1 inhibition signature in HSC-3 cells and interrogated it in TCGA HNSCC. We then examined the effects of inhibition of β-catenin-CBP interaction with E7386 on DPAGT1 expression using ChIP-seq and computational approaches. E7386, a novel β-catenin/CBP modulator displays activity profile that closely overlaps with that of ICG-001, but exhibits ~50 - 100-fold lower EC50 values. Ectopic expression of DPAGT1 in indolent CAL27 cells induced epithelial-to-mesenchymal transition (EMT) which coincided with increased abundance of active β-catenin. Partial knockdown of DPAGT1 with siRNA in metastatic HSC-3 cells inhibited EMT, diminished cell migration and enhanced intercellular adhesion. Inhibition of the DPAGT1 enzyme activity using tunicamycin interfered with orthotopic tongue tumor growth and metastasis. DPAGT1 knockdown in HSC-3 cells defined DPAGT1-activated gene signature as enriched in pro-tumorigenic signaling pathways, including stem cell-like genes. Integrative analysis of the DPAGT1-activated genes in TCGA validated the association of DPAGT1 activity with the EMT transcription factors, ZEB1, Twist1/2 and Slug. ChIP-seq analyses without and with the E7386 treatment revealed reduced occupancy of H3K4me3 at two DPAGT1 transcription start sites following the E7386 treatment. In conclusion, our studies align aberrant activation of DPAGT1 with the induction of EMT and stem cell associated genes and suggest a novel role of β-catenin/CBP/MLL1 in the epigenetic regulation DPAGT1 and protein N-glycosylation in HNSCC. Citation Format: Khalid A. Alamoud, Vinay Kartha, Huamei Yang, Andrew Tilston-Lunel, Anthony Federico, Manish Bais, Takachi Owa, Kenichi Nomoto, Xalarabos Varelas, Stefano Monti, Maria A. Kukuruzinska. Identification of a novel role for the β-catenin/CBP signaling in epigenetic regulation of the N-glycosylation gene, DPAGT1, in head and neck cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2442.

Sex disparities in head & neck cancer driver genes: An analysis of the TCGA dataset.

Survival in head and neck squamous cell carcinoma (HNSCC) has been associated with patient sex, typically with males experiencing poorer outcomes. It is unclear if this disparity is based in divergent tumor biology. We analyzed the TCGA HNSCC cohort to uncover disparities in the somatic single nucleotide variation (SNV), copy number alteration (CNA) and mRNA abundance profiles between males and females. Critically, we stratified our results by tumor HPV status to control for this significant confounder. SNV, CNA and mRNA abundance differences between males and females were compared separately for the HPV-positive (n=67) and negative (n=431) TCGA HNSCC cohorts. Overall survival outcomes were compared in males and females in both HPV-positive and HPV-negative subsets of patients. Females were found to have poorer overall survival than males (p=0.048), largely due to higher rates of HPV-positive disease among men. SNV analysis revealed that in HPV-positive disease, there were no differences by sex after accounting for the false discovery rate (FDR). In HPV-negative tumors, BRWD3 mutations occurred more frequently in the tumors of female patients compared to males after adjusting for the FDR (p=0.02). Further, HPV-negative BRWD3 mutant tumors were found to have significantly worse 5-year overall survival compared to wildtype on multivariate analysis (p=0.02). There were 88 heterozygous deletions and 14 amplifications that were differentially altered between male and female HPV-negative tumors and associated with expression changes. Pathway analysis of these genes revealed that tumors from males were enriched in five pathways including chemokine and phosphophatidylinositol signaling. Reanalysis of the TCGA HNSCC dataset stratified by sex revealed that males in this cohort had a significant survival advantage, due to a higher proportion of HPV-positive disease. Mutations in BRWD3 were more frequent in HPV-negative tumors of females and were associated with poorer overall survival. BRWD3 may represent a novel biomarker of patient outcomes, but will require additional validation.

Identification and validation of an alternative splicing-based prognostic signature for head and neck squamous cell carcinoma.

Increasing evidence has demonstrated that changes in alternative splicing (AS) events are closely associated with the initiation and progression of cancer. However, the concrete role of AS in tumorigenesis of head and neck squamous cell carcinoma (HNSCC) is poorly known. In this study, we aimed to investigate the AS profile in HNSCC, and build up a robust AS-based prognostic signature for HNSCC. Our results revealed a total of 4068 overall survival (OS) associated AS events in the TCGA HNSCC cohort. The whole TCGA HNSCC cohort was randomly divided into discovery cohort and validation cohort. A prognostic signature including five AS events was developed with the discovery cohort based on the most significant OS-associated AS events. Then it was further successfully validated in the validation cohort. The AS-based risk signature was an independent prognostic indicator in both discovery cohort and validation cohort. This prognostic signature-based nomogram model showed excellent performance for predicting the OS of HNSCC. Splicing network analysis have identified the most correlated splicing factor-AS network in HNSCC. Collectively, we have constructed a robust AS-based prognostic signature which might contribute to improve the clinical outcome of HNSCC.

Identification of Immune-Desert Phenotypes in Head and Neck Squamous Cell Carcinoma With a Nine-Gene Signature Prediction Model Using Bioinformatics&amp;nbsp;Methods

Background: Research into anti-programmed cell death-1 and anti-programmed cell death ligand-1(anti-PD-1/PD-L1) immunotherapy has increased exponentially in the past years. However, immune check inhibitor (ICI) therapies are not effective in all patients. In this study, we explored biomarkers within the TME to identify specific immunophenotypes, and to predict the efficacy of immunotherapy in patients with head and neck squamous cell carcinoma (HNSCC) through bioinformatics analysis. Method: Data on HNSCC patients were derived from TCGA and GEO databases and re-annotated through BLAST analysis. The composition of immune cells in HNSCC was estimated by CIBERSORT and ssGSEA. Weighted correlation network analysis (WGCNA) was used to measure co-expression relationships. The Lasso-logistic model was used to screen biomarkers associated with specific cancer immunophenotypes. A bootstrapping algorithm was used to select optimal cut-off values. Gene function analysis was performed using the Reactome pathway databases. The ESTIMATE algorithm was used to infer the tumor purity. Target drugs which might be used in the immune-desert patient population were screened by a connectivity mapping analysis. Findings: A high degree of similarity in the genes and pathways enriched from the immune-related modules of TCGA HNSCC and GSE65858 datasets was observed. Sixty hub genes were identified through the intersection of two modules to distinguish between immune-desert and non-immune-desert phenotypes. Furthermore, a nine-gene signature prediction model (ALOX5, ARHGDIB, GIMAP1, ITGAL, MAP4K1, RASSF2, SH2D1A, SLAMF6, UCP2) was selected which had a prediction accuracy of 83%, and was related to the efficacy of ICI therapies. Finally, it was found that at least five of these nine genes with negative expression could predict the tumor samples as immune-desert phenotype, and protein kinase C (PKC) activators might be useful drugs for immune-desert patient population in HNSCC. Interpretation: Our nine-gene model may predict the immunophenotype in HNSCC. At least five of these nine genes with negative expression could distinguish immune-desert phenotypes. These findings provide a new strategy to identify the immune state of the TME in HNSCC, and the nine genes may serve as potential biomarkers for clinical applications. The PKC activators might be a potential treatment for immune-desert phenotype tumor. Funding Statement: This work was supported by The National Key Research and Development Program of China (2017YFC1308900), Technological Special Project of Liaoning Province of China (2019020176-JH1/103), Science and Technology Project of Liaoning Province (NO.2013225585), The General Projects of Liaoning Province Colleges and Universities (LFWK201706), National Natural Science Foundation of China (NO.81302023) and China Medical University Youth Backbone Support Plan Project (QGZ2018047). Declaration of Interests: The authors declare no conflict of interests.

Smoking and gender modify the effect of TWIST on patient survival in head and neck squamous carcinoma.

PurposeTWIST is a critical factor for predicting prognosis in several human cancers. Here, we study the prognostic significance of TWIST1 and TWIST2 in Head and Neck squamous cell carcinoma (HNSCC) as well as interactions of TWISTs with both gender and smoking in patient survival.Methodsupper quartile normalized RNA-seq V2 RSEM values of TWIST1 and TWIST2 expressions were retrieved from a TCGA HNSCC dataset. Kaplan-Meier survival curves were used to assess the associations of TWIST1 and TWIST2 with patient survival, and multivariate Cox proportional hazards regression models were used to estimate the hazards ratios (HRs) and their 95% confidence intervals (CIs).ResultsSurvival analyses showed that high TWIST1 expression was associated with a poor overall survival at a borderline significance level, while a superior but not statistically significant overall survival was observed in high TWIST2 expression. The multivariate Cox proportional hazards regression model showed a significantly elevated risk of death (HR=1.37, p = 0.038) in patients with high TWIST1 compared to low TWIST1, and a borderline significantly decreased risk of death (HR = 0.74, p = 0.055) in patients with high TWIST2 compared to low TWIST2. Further stratification analyses showed that increased risks of death were found significantly in male and borderline significantly in smoker patients with high TWIST1 compared to low one, and a significantly decreased risk of death in non-smoker patients with high TWIST2 compared to low one.ConclusionsTWIST1 and TWIST2 are differentially associated with HNSCC patient survival. Gender and smoking could modify the effect of TWISTs on the risk of death in HNSCC patients.

FSCN1 is upregulated by SNAI2 and promotes epithelial to mesenchymal transition in head and neck squamous cell carcinoma.

In this study, we investigated whether there is any association between the expression of FSCN1 and SNAI2 and the possible underlying mechanisms in head and neck squamous cell carcinoma (HNSC). In addition, we also investigated whether FSCN1 modulates epithelial-to-mesenchymal transition (EMT) in HNSC cells. Microarray data of dysregulated genes in HNSC were searched in GEO datasets. The association between FSCN1 expression and the 5-year/10-year overall survival (OS), as well as the correlation between the expression of FSCN1 and SOX2, MYBL2, SNAI2, STAT1, and SOX4, was analyzed based on data in TCGA HNSC cohort (TCGA-HNSC). The binding site of SNAI2 in FSCN1 promoter was verified using luciferase reporter assay. SCC9 and SCC15 cells were transfected with pCMV-SNAI2 or pCMV-FSCN1 expression vector or the empty control. Alteration of E-cadherin, Claudin 1, Vimentin, and N-cadherin was then quantified. Our results showed that FSCN1 is significantly upregulated in HNSC tissues compared with the normal control tissues. High FSCN1 expression is associated with worse 5-year and 10-year OS among the HNSC patients. Bioinformatic prediction showed a highly possible SNAI2 binding site in FSCN1 promoter and following luciferase reporter assay verified this site. SNAI2 overexpression significantly increased FSCN1 expression at both mRNA and protein level. FSCN1 overexpression reduced the expression of E-cadherin and Claudin 1, but increased the expression of Vimentin and N-cadherin in SCC9 and SCC-15 cells. Therefore, we infer that FSCN1 is a downstream effector of SNAI2 in promoting EMT in HNSC cells.