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Hypopharyngeal carcinoma is one of the malignant tumors of the head and neck with a particularly poor prognosis. Recurrence and metastasis are important reasons for poor prognosis of hypopharyngeal cancer patients, and malignant proliferation, migration, and invasion of tumor cells are important factors for recurrence and metastasis of hypopharyngeal cancer. Therefore, elucidating hypopharyngeal cancer cells' proliferation, migration, and invasion mechanism is essential for improving diagnosis, treatment, and prognosis. Plasmacytoma Variant Translocation 1 (PVT1) is considered a potential diagnostic marker and therapeutic target for tumors. However, it remains unclear whether PVT1 is related to the occurrence and development of hypopharyngeal cancer and its specific mechanism. In this study, the promoting effect of PVT1 on the proliferation, migration, and invasion of hypopharyngeal carcinoma FaDu cells was verified by cell biology experiments and animal studies, and it was found that PVT1 inhibited the expression of TGF-β, suggesting that PVT1 may regulate the occurrence and development of hypopharyngeal carcinoma FaDu cells through TGF-β., (© 2024. The Author(s).)

Aims: Hypopharyngeal cancer (HPC) is associated with the worst prognosis of all head and neck cancers and is typically identified in an advanced stage at the time of diagnosis. While oxidative stress might contribute to the onset of HPC in patients using tobacco or alcohol, the extent of this influence and the characteristics of HPC cells in advanced stage remain to be investigated. In this study, we explored whether HPC cells survived from necrotic xenograft tumors at late stage would display increased tumor resistance along with altered tolerance to oxidative stress. Results: The remnant living HPC cells isolated from a late-stage xenograft tumor, named FaDu ex vivo cells, showed stronger chemo- and radioresistance, tumorigenesis, and invasiveness compared with parental FaDu cells. FaDu ex vivo cells also displayed increased angiogenic ability after re-transplantation in mice visualized by in vivo near infrared-II fluorescence imaging modality. Moreover, FaDu ex vivo cells exhibited significant tumor-initiating cell (TIC)-related properties accompanied by a reduction of the level of reactive oxygen species, which was associated with the upregulation of transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). Interestingly, inhibition of Nrf2 by the RNA interference and the chemical inhibitor could reduce the TIC-related properties of FaDu ex vivo cells. Innovation: Oxidative stress potentially initiates HPC, but elevation of Nrf2-associated antioxidant mechanisms would be essential to mitigate this effect for promoting and sustaining the stemness of HPC at the advanced stage. Conclusion: Present data suggest that the antioxidant potency of advanced HPC would be a therapeutic target for the design of adjuvant treatment. Antioxid. Redox Signal. 41, 505-521.

Previous studies have indicated that transmembrane protein 16A (TMEM16A) plays a crucial role in the pathogenesis and progression of various tumors by influencing multiple signaling pathways. However, the role of TMEM16A in regulating autophagy via the mammalian target of rapamycin (mTOR) pathway and its impact on the development of hypopharyngeal squamous cell carcinoma (HSCC) remain unclear. Immunohistochemistry and western blotting were used to assess the expression of TMEM16A in HSCC tissues and metastatic lymph nodes. Manipulation of TMEM16A expression levels was achieved in the FaDu cell line through overexpression or knockdown, followed by assessment of its biological effects using cell colony formation, wound healing, transwell and invasion assays. Additionally, apoptosis and autophagy-related proteins, as well as autophagosome formation, were evaluated through western blotting, transmission electron microscopy and immunofluorescence following TMEM16A knockdown or overexpression in FaDu cells. Our study revealed significantly elevated levels of TMEM16A in both HSCC tissues and metastatic lymph nodes compared with normal tissues. In vitro experiments demonstrated that silencing TMEM16A led to a notable suppression of HSCC cell proliferation, invasion and migration. Furthermore, TMEM16A silencing effectively inhibited tumor growth in xenografted mice. Subsequent investigations indicated that knockdown of TMEM16A in HSCC cells could suppress mTOR activation, thereby triggering autophagic cell death by upregulating sequestosome-1 (SQSTM1/P62) and microtubule-associated protein light chain 3 II (LC3II). This study highlights the crucial role of TMEM16A in modulating autophagy in HSCC, suggesting its potential as a therapeutic target for the treatment of this malignancy., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Hypopharyngeal squamous cell carcinoma (HSCC) is a malignant tumor of head and neck. It was verified that circ0005027 was downregulated in HSCC tissues. Here, we aimed to investigate the function and specific regulatory mechanism of circ0005027 in HSCC. Ten pairs tissues of HSCC and adjacent para-cancer were collected. Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) measured circ0005027, miR-548c-3p, and Cadherin 1 (CDH1) mRNA expression. CCK-8 analyzed cell proliferation viability. Flow cytometry assay detected cell cycle and apoptosis rate. Clonal formation assay measured the clonal ability. Transwell detected cell invasion ability. Western blot was performed to detect CDH1, LAST1, p-LAST1, MST1, p-MST1, YAP1, p-YAP1, TAZ and p-TAZ protein level. Dual-luciferase, RIP and RNA pull-down assay identified the target relationship among circ0005027, miR-548c-3p and CDH1. circ0005027 was decreased in tissues and FaDu cells of HSCC. Overexpression of circ0005027 inhibited cell viability, G1-S transition, clonal formation, and invasion and increased cell apoptosis. circ0005027 acted as a ceRNA and decreased circ0005027 enhanced the malignant process of FaDu cells through sponging miR-548c-3p and inhibiting CDH1 expression. Overexpression of CDH1 activated YAP1/TAZ pathway and inhibited the growth of HSCC in vitro. circ0005027 might act as a potential biomarker for the progression and prognosis prediction in HSCC by regulating miR-548c-3p/CDH1/ YAP1/TAZ signaling pathway., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Hypopharyngeal cancer is a disease that is associated with EGFR-mutated lung adenocarcinoma. Here we utilized a bioinformatics approach to identify genetic commonalities between these two diseases. To this end, we examined microarray datasets from GEO (Gene Expression Omnibus) to identify differentially expressed genes, common genes, and hub genes between the selected two diseases. Our analyses identified potential therapeutic molecules for the selected diseases based on 10 hub genes with the highest interactions according to the degree topology method and the maximum clique centrality (MCC). These therapeutic molecules may have the potential for simultaneous treatment of these diseases., (© 2024 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

To explore the hub comorbidity genes and potential pathogenic mechanisms of hypopharyngeal carcinoma with esophageal carcinoma, and evaluate their diagnostic value for hypopharyngeal carcinoma with co-morbid esophageal carcinoma. We performed gene sequencing on tumor tissues from 6 patients with hypopharyngeal squamous cell carcinoma with esophageal squamous cell carcinoma (hereafter referred to as "group A") and 6 patients with pure hypopharyngeal squamous cell carcinoma (hereafter referred to as "group B"). We analyzed the mechanism of hub genes in the development and progression of hypopharyngeal squamous cell carcinoma with esophageal squamous cell carcinoma through bioinformatics, and constructed an ROC curve and Nomogram prediction model to analyze the value of hub genes in clinical diagnosis and treatment. 44,876 genes were sequenced in 6 patients with group A and 6 patients with group B. Among them, 76 genes showed significant statistical differences between the group A and the group B.47 genes were expressed lower in the group A than in the group B, and 29 genes were expressed higher. The top five hub genes were GABRG2, CACNA1A, CNTNAP2, NOS1, and SCN4B. GABRG2, CNTNAP2, and SCN4B in the hub genes have high diagnostic value in determining whether hypopharyngeal carcinoma patients have combined esophageal carcinoma (AUC: 0.944, 0.944, 0.972). These genes could possibly be used as potential molecular markers for assessing the risk of co-morbidity of hypopharyngeal carcinoma combined with esophageal carcinoma., (© 2024. The Author(s).)

Background: Advances in upper gastrointestinal endoscopic technology have enabled early detection and treatment of hypopharyngeal cancer. However, in-depth pharyngeal observations require sedation and are invasive. It is important to establish a minimally invasive and simple evaluation method to identify high-risk patients., Methods: Eighty-seven patients with superficial hypopharyngeal cancer and 51 healthy controls were recruited. We assessed the methylation status of DCC, PTGDR1, EDNRB, and ECAD, in tissue and saliva samples and verified the diagnostic accuracy by methylation analyses of their promoter regions using quantitative methylation-specific PCR., Results: Significant differences between cancer and their surrounding non-cancerous tissues were observed in the methylation values of DCC (p = 0.003), EDNRB (p = 0.001), and ECAD (p = 0.043). Using receiver operating characteristic analyses of the methylation values in saliva samples, DCC showed the highest area under the curve values for the detection of superficial hypopharyngeal cancer (0.917, 95% confidence interval = 0.864-0.970), compared with those for EDNRB (0.680) and ECAD (0.639). When the cutoff for the methylation values of DCC was set at ≥0.163, the sensitivity to detect hypopharyngeal cancer was 82.8% and the specificity was 90.2%., Conclusions: DCC methylation in saliva samples could be a non-invasive and efficient tool for early detection of hypopharyngeal cancer in high-risk patients., (© 2024. The Author(s).)

Hypopharyngeal squamous cell carcinoma (HSCC) is a kind of malignant tumor with a poor prognosis and low quality of life in the otolaryngology department. It has been found that microRNA (miRNA) plays an important role in the occurrence and development of various tumors. This study found that the expression level of miRNA-107 (miR-107) in HSCC was significantly reduced. Subsequently, we screened out the downstream direct target gene Neuronal Vesicle Trafficking Associated 1 (NSG1) related to miR-107 through bioinformatics analysis and found that the expression of NSG1 was increased in HSCC tissues. Following the overexpression of miR-107 in HSCC cells, it was observed that miR-107 directly suppressed NSG1 expression, leading to increased apoptosis, decreased proliferation, and reduced invasion capabilities of HSCC cells. Subsequent experiments involving the overexpression and knockdown of NSG1 in HSCC cells demonstrated that elevated NSG1 levels enhanced cell proliferation, migration, and invasion, while the opposite effect was observed upon NSG1 knockdown. Further investigations revealed that changes in NSG1 levels in the HSCC cells were accompanied by alterations in ERK signaling pathway proteins, suggesting a potential regulatory role of NSG1 in HSCC cell proliferation, migration, and invasion through the ERK pathway. These findings highlight the significance of miR-107 and NSG1 in hypopharyngeal cancer metastasis, offering promising targets for therapeutic interventions and prognostic evaluations for HSCC.

Background: The cellular origin of hypopharyngeal diseases is crucial for further diagnosis and treatment, and the microenvironment in tissues may also be associated with specific cell types at the same time. Normal adjacent tissues (NATs) of hypopharyngeal carcinoma differ from non-tumor-bearing tissues, and can influenced by the tumor. However, the heterogeneity in kinds of disease samples remains little known, and the transcriptomic profile about biological information associated with disease occurrence and clinical outcome contained in it has yet to be fully evaluated. For these reasons, we should quickly investigate the taxonomic and transcriptomic information of NATs in human hypopharynx., Results: Single-cell suspensions of normal adjacent tissues (NATs) of hypopharyngeal carcinoma were obtained and single-cell RNA sequencing (scRNA-seq) was performed. We present scRNA-seq data from 39,315 high-quality cells in the hypopharyngeal from five human donors, nine clusters of normal adjacent human hypopharyngeal cells were presented, including epithelial cells, endothelial cells (ECs), mononuclear phagocyte system cells (MPs), fibroblasts, T cells, plasma cells, B cells, mural cells and mast cells. Nonimmune components in the microenvironment, including epithelial cells, endothelial cells, fibroblasts and the subpopulations of them were performed., Conclusions: Our data provide a solid basis for the study of single-cell landscape in human normal adjacent hypopharyngeal tissues biology and related diseases., (© 2024. The Author(s).)

Introduction: Hypopharyngeal squamous cell carcinoma (HSCC) is one of the malignant tumors with the worst prognosis in head and neck cancers. The transformation from normal tissue through low-grade and high-grade intraepithelial neoplasia to cancerous tissue in HSCC is typically viewed as a progressive pathological sequence typical of tumorigenesis. Nonetheless, the alterations in diverse cell clusters within the tissue microenvironment (TME) throughout tumorigenesis and their impact on the development of HSCC are yet to be fully understood., Methods: We employed single-cell RNA sequencing and TCR/BCR sequencing to sequence 60,854 cells from nine tissue samples representing different stages during the progression of HSCC. This allowed us to construct dynamic transcriptomic maps of cells in diverse TME across various disease stages, and experimentally validated the key molecules within it., Results: We delineated the heterogeneity among tumor cells, immune cells (including T cells, B cells, and myeloid cells), and stromal cells (such as fibroblasts and endothelial cells) during the tumorigenesis of HSCC. We uncovered the alterations in function and state of distinct cell clusters at different stages of tumor development and identified specific clusters closely associated with the tumorigenesis of HSCC. Consequently, we discovered molecules like MAGEA3 and MMP3, pivotal for the diagnosis and treatment of HSCC., Discussion: Our research sheds light on the dynamic alterations within the TME during the tumorigenesis of HSCC, which will help to understand its mechanism of canceration, identify early diagnostic markers, and discover new therapeutic targets., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Tie, Zhu, Yu, Dou, Wang, Wang and Ni.)

Hypopharyngeal squamous cell carcinoma (HPSCC) has the worst prognosis among head and neck squamous cell carcinomas. The lack of available tumor cell lines poses a significant obstacle to the development of efficient treatments for HPSCC. In this study, we successfully established a novel cell line, named CZH1, from the postcricoid region of a Chinese male patient with a T3N0M0 HPSCC. Short tandem repeat analysis confirmed the uniqueness of CZH1. The cell line was characterized by its phenotypes, biomarkers, and genetics. Importantly, CZH1 cells retained the typical features of epithelial malignancy, similar to the primary tumor tissue. Furthermore, CZH1 demonstrated a greater capacity for invasion and increased susceptibility to irradiation in comparison to FaDu, which is the most commonly used HPSCC cell line. Whole-exome sequencing analysis revealed that CZH1 cells had typical genomic features of HNSCC, including mutations of TP53 and amplifications of multiple transcripts. Therefore, our newly developed CZH1 cell line could serve as an efficient tool for the in vitro investigation of the etiology, pathogenesis, and preclinical treatment of HPSCC., (© 2024. The Author(s) under exclusive licence to Japan Human Cell Society.)

Hypopharyngeal carcinoma is the most malignant type of head and neck squamous cell carcinoma, and lncRNAs play an important role in its formation and progression. However, the related specific mechanisms are rarely studied. lncRNAs closely associated with hypopharyngeal cancer were examined by lncRNA sequencing for in-depth exploration of the relationship between HOXC-AS2 and hypopharyngeal cancer pathogenesis. The mRNA expression of HOXC-AS2 and related genes was measured by qRT-PCR, and the biological function of HOXC-AS2 in hypopharyngeal carcinoma was demonstrated by gain- and loss-of-function experiments. RNA pulldown, RNA immunoprecipitation (RIP) and gene body truncation experiments and transcriptome sequencing were used to investigate the potential mechanism of HOXC-AS2 and its downstream genes, including P62, NF-KB and HMOX1. Finally, the biological function of HOXC-AS2 was confirmed in animal experiments. HOXC-AS2 and P62 expression was significantly upregulated in hypopharyngeal cancer tissues compared with normal hypopharyngeal tissues, while HMOX1 expression was decreased. Functionally, HOXC-AS2 overexpression can promote the viability, proliferation, migration and invasion of hypopharyngeal cancer cells and facilitate hypopharyngeal cancer progression. It was confirmed that HOXC-AS2 can bind to the P62 protein and activate the NF-KB signaling pathway, thereby affecting HMOX1 expression and regulating autophagy in hypopharyngeal cancer cells, ultimately regulating the formation and progression of hypopharyngeal cancer. In conclusion, our findings suggest that HOXC-AS2 regulates the progression of hypopharyngeal cancer by regulating autophagy and is abnormally highly expressed in hypopharyngeal cancer tissues. HOXC-AS2 may become a new target for the diagnosis and treatment of hypopharyngeal cancer.

Background: Hypopharyngeal squamous cell carcinoma (HPSCC) has the worst prognosis among all head-and-neck cancers, and treatment options are limited. Tumor microenvironment (TME) analysis can help identify new therapeutic targets and combined treatment strategies., Methods: Six primary HPSCC tissues and two adjacent normal mucosae from six treatment-naïve patients with HPSCC were analyzed using scRNA-seq. Cell types were curated in detail, ecosystemic landscapes were mapped, and cell-cell interactions were inferred. Key results were validated with The Cancer Genome Atlas and cell biology experiments., Results: Malignant HPSCC epithelial cells showed significant intratumor heterogeneity. Different subtypes exhibited distinct histological features, biological behaviors, and spatial localization, all affecting treatment selection and prognosis. Extracellular matrix cancer-associated fibroblasts (mCAFs) expressing fibroblast activation protein were the dominant CAFs in HPSCC tumors. mCAFs, constituting an aggressive CAF subset, promoted tumor cell invasion, activated endothelial cells to trigger angiogenesis, and synergized with SPP1 + tumor associated macrophages to induce tumor progression, ultimately decreasing the overall survival of patients with HPSCC. Moreover, the LAMP3 + dendritic cell subset was identified in HPSCC and formed an immunosuppressive TME by recruiting Tregs and suppressing CD8+ T cell function., Conclusions: mCAFs, acting as the communication center of the HPSCC TME, enhance the invasion ability of HPSCC cells, mobilizing surrounding cells to construct a tumor-favorable microenvironment. Inhibiting mCAF activation offers a new anti-HPSCC therapeutic strategy. Video Abstract., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Objective: MicroRNA-29a-3p has been reported in a variety of cancers, but its role in hypopharyngeal cancer remains unclear. This study was to determine the role of microRNA-29a-3p in the occurrence and development of hypopharyngeal cancer., Methods: 40 patients with hypopharyngeal cancer who underwent surgery in the Affiliated Hospital of Jining Medical University from April 2013 to November 2017 were selected for this study. The cancer tissue samples of the patients were collected, and the patients were followed up for three years. The expression of microRNA-29a-3p in tissue samples was detected by in situ hybridization with fluorescent probe, and the relationships among microRNA-29a-3p and clinicopathological factors, postoperative recurrent-metastasis, survival time were studied. Immunohistochemical was used to detect the expression of Ki67 and E-cadherin in tissue samples., Results: Combined with HE staining results showed that microRNA-29a-3p expression was relatively high in non-cancer tissue cells (red blood cells and fibroblasts in tumor interstitial vessels), but was relatively low in cancer tissue and cells. According to the follow-up data of 40 patients with hypopharyngeal cancer, tumor size, T-stage, tumor differentiation, postoperative recurrent-metastasis of hypopharyngeal cancer patients were significantly negatively correlated with microRNA-29a-3p (p < 0.05). Immunohistochemica results further confirmed that microRNA-29a-3p was negatively correlated with the expression of Ki67 and E-cadherin. The survival time of patients positively related with microRNA-29a-3p expression (p < 0.05). Moreover, ROC curve analysis showed that the area under the curve of the combined detection of miRNA-29a-3p+Ki67+E-cadherin was larger than that of the single detection of the three indexes., Conclusions: The expression of microRNA-29a-3p is closely related to the occurrence, development and prognosis of hypopharyngeal cancer, and it affects the proliferation and invasion. This indicates that microRNA-29a-3p serves as a therapeutic target for the occurrence and development of hypopharyngeal cancer. The evidence of study designs of this study is IV using "Oxford Centre for Evidence-Based Medicine 2011 Levels of Evidence"., (Copyright © 2023 Associação Brasileira de Otorrinolaringologia e Cirurgia Cérvico-Facial. Published by Elsevier España, S.L.U. All rights reserved.)

Hypopharyngeal cancer is a subtype of the head and neck malignancies. We aimed to explore the role of lysine-specific demethylase 1 (LSD1/KDM1A) in the progression of hypopharyngeal cancer and to identify the potential mechanisms. First, LSD1 expression in head and neck squamous cell carcinoma (HNSCC) tissues and the correlation between LSD1 and the stage of HNSC were analyzed by the University of ALabama at Birmingham CANcer data analysis Portal (UALCAN). Following LSD1 silencing, proliferation of pharyngeal cancer cell line FaDu cells was evaluated by cell counting kit-8 and colony formation assays. Wounding healing and transwell assays were used to measure the capacities of migration and invasion. In addition, expression of proteins related to epithelial-to-mesenchymal transition (EMT), autophagy, and pyroptosis was tested by Western blot analysis or immunofluorescence. After treatment with autophagy inhibitor 3-methyladenine (3-MA) or NLR family pyrin domain containing 3 (NLRP3) inhibitor MCC950, the malignant biological properties were measured again. High LSD1 expression was observed in HNSC tissues, which was correlated with stage. LSD1 knockdown significantly suppressed the proliferation, migration, invasion, and EMT of hypopharyngeal cancer cells. Moreover, autophagy and pyroptosis were induced by LSD1 depletion, observed by the enhanced fluorescence intensity of LC3, gasdermin-D (GSDMD)-N, and apoptosis-associated speck-like protein containing a CARD (ASC), accompanied by upregulated expression of LC3II/LC3I, Beclin-1, NLRP3, cleaved-caspase 1, ASC, interleukin (IL)-1β, and IL-18 and downregulated expression of p62. Importantly, 3-MA or MCC950 addition obviously reversed the inhibitory effects of LSD1 silencing on the proliferation, migration, invasion, and EMT of hypopharyngeal cancer cells. To sum up, LSD1 silencing could restrain the progression of hypopharyngeal cancer cells by inducing autophagy and pyroptosis., Competing Interests: None

The efficacy of the first-line treatment for hypopharyngeal carcinoma (HPC), a predominantly male cancer, at advanced stage is only about 50% without reliable molecular indicators for its prognosis. In this study, HPC biopsy samples collected before and after the first-line treatment are classified into different groups according to treatment responses. We analyze the changes of HPC tumor microenvironment (TME) at the single-cell level in response to the treatment and identify three gene modules associated with advanced HPC prognosis. We estimate cell constitutions based on bulk RNA-seq of our HPC samples and build a binary classifier model based on non-malignant cell subtype abundance in TME, which can be used to accurately identify treatment-resistant advanced HPC patients in time and enlarge the possibility to preserve their laryngeal function. In summary, we provide a useful approach to identify gene modules and a classifier model as reliable indicators to predict treatment responses in HPC., (© 2023. The Author(s).)

Hypopharyngeal carcinoma is a cancer with the worst prognosis. We constructed the first single-cell transcriptome map for hypopharyngeal carcinoma and explored its underlying mechanisms. We systematically studied single-cell transcriptome data of 17,599 cells from hypopharyngeal carcinoma and paracancerous tissues. We identified categories of cells by dimensionality reduction and performed further subgroup analysis. Focusing on the potential mechanism in the cellular communication of hypopharyngeal carcinoma, we predicted ligand-receptor interactions and verified them via immunohistochemical and cellular experiments. In total, seven cell types were identified, including epithelial and myeloid cells. Subsequently, subgroup analysis showed significant tumor heterogeneity. Based on the pathological type of squamous cell carcinoma, we focused on intercellular communication between epithelial cells and various cells. We predicted the crosstalk and inferred the regulatory effect of cellular active ligands on the surface receptor of epithelial cells. From the top potential pairs, we focused on the BMPR2 receptor for further research, as it showed significantly higher expression in epithelial cancer tissue than in adjacent tissue. Further bioinformatics analysis, immunohistochemical staining, and cell experiments also confirmed its cancer-promoting effects. Overall, the single-cell perspective revealed complex crosstalk in hypopharyngeal cancer, in which BMPR2 promotes its proliferation and migration, providing a rationale for further study and treatment of this carcinoma., (© 2022. The Author(s).)

The research on targeted therapy of hypopharyngeal cancer is very scarce. The discovery of new targeted driver genes will promote the progress of hypopharyngeal cancer therapy to a great extent. In our research, whole-exome sequencing in 10 patients with hypopharyngeal cancer was performed to identify single nucleotide variations (SNVs) and insertions and deletions (INDELs). American College of Medical Genetics and Genomics (ACMG) guidelines were used to evaluate the pathogenicity of the selected variants. 8113 mutation sites in 5326 genes were identified after strict screening. We identified 72 pathogenic mutations in 53 genes according to the ACMG guidelines. Gene Ontology (GO) annotation and KEGG enrichment analysis show the effect of these genes on cancer. Protein-protein interaction (PPI) was analyzed by string online software. The validation results of the ualcan database showed that 22 of the 53 genes may be related to the poor prognosis of patients with hypopharyngeal cancer. RBM20 has the most significant correlation with hypopharyngeal cancer, and it is likely to be the driver gene of hypopharyngeal cancer. In conclusion, we found possible therapeutic targets for hypopharyngeal cancer, especially RBM20 and KMT2C. Our study provides a basis for the pathogenesis and targeted therapy of hypopharyngeal cancer., (© 2023. The Author(s).)

Background: The patients with dual oesophageal squamous cell carcinoma (ESCC) and hypopharyngeal cancer (HPC) have poor prognosis; their underlying genetic pathogenesis is unclear. We hypothesise that development of synchronous ESCC/HPC depends on multicentricity or independent origin, rather than multifocality due to local or lateral spreading., Method: Multiple region whole-exome sequencing (M-WES) and clonality analysis were used to assess clonal relationship and spatial inter- or intra-tumour heterogeneity (ITH) in 62 tumour regions from eight dual ESCC/HPC and ten ESCC patients., Results: All synchronous ESCC/HPC patients had COSMIC 16 mutation signatures, compared to only 40% ESCC in the current study (p = 0.013) and public data set (n = 165, p = 0.003). This alcohol consumption-related mutation signature 16, commonly involved in multiple alcohol-related cancers, was significantly associated with drinking and alcohol metabolism-related ADH1B rs1229984. The mutational landscape and copy number profiles were completely distinct between the two primary tumours; clonality analysis further suggested the two primary tumours shared no or only one clone accompanying independent subclone evolution. M-WES strategy demonstrated higher sensitivity and accuracy for detection of mutational prevalence and the late branch mutations among different regions in the ESCC tumours, compared to traditional sequencing analysis based on single biopsy strategy. Patients with high ITH assessed by cancer cell fraction analysis after M-WES were significantly associated with both relapse and survival., Conclusions: Our hypothesis-generating M-WES ITH assessment data have implications for prognostication. Collectively, our findings support multicentric independent clonal evolution, the field cancerisation theory, and suggest novel insights implicating an aetiologic role of alcohol metabolism in dual ESCC/HPC carcinogenesis., (© 2022. The Author(s).)

Several members of the transmembrane protein family are associated with the biological processes of human malignancies; however, the expression pattern and biological function of one family member, TMEM184B, in hypopharyngeal squamous cell carcinoma (HPSCC) are not fully understood. The expression between HPSCC tumours and adjacent normal tissues was determined by the Immunohistochemistry (IHC). A bioinformatics analysis was performed to verify the expression pattern of TMEM184B in The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Furthermore, in vitro assays on cell proliferation, invasion, migration and in vivo experiments on tumour growth and apoptosis of TMEM184B in HPSCC were performed. We found that the HPSCC tissues had a significantly higher expression of TMEM184B than the adjacent normal tissues. Bioinformatics analysis confirmed the different expression of TMEM184B expression in HPSCC. Furthermore, in vitro and in vivo experiments demonstrated that TMEM184B promotes HPSCC cell growth, cell invasion and migration in FaDu cells, whereas flow cytometry assay showed that TMEM184B inhibited cell apoptosis. Our study revealed for the first time that TMEM184B might serve an oncogenic function in HPSCC and could be a potential diagnostic biomarker and therapeutic target for HPSCC., (© 2022 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

CD271 (also referred to as nerve growth factor receptor or p75 NTR ) is expressed on cancer stem cells in hypopharyngeal cancer (HPC) and regulates cell proliferation. Because elevated expression of CD271 increases cancer malignancy and correlates with poor prognosis, CD271 could be a promising therapeutic target; however, little is known about the induction of CD271 expression and especially its promoter activity. In this study, we screened transcription factors and found that RELA (p65), a subunit of nuclear factor kappaB (NF-κB), is critical for CD271 transcription in cancer cells. Specifically, we found that RELA promoted CD271 transcription in squamous cell carcinoma cell lines but not in normal epithelium and neuroblastoma cell lines. Within the CD271 promoter sequence, region + 957 to + 1138 was important for RELA binding, and cells harboring deletions in proximity to the + 1045 region decreased CD271 expression and sphere-formation activity. Additionally, we found that clinical tissue samples showing elevated CD271 expression were enriched in RELA-binding sites and that HPC tissues showed elevated levels of both CD271 and phosphorylated RELA. These data suggested that RELA increases CD271 expression and that inhibition of RELA binding to the CD271 promoter could be an effective therapeutic target., (© 2022. The Author(s).)

Objective: Follistatin (FST) inhibits the action of activin by interfering with the binding of activin to its receptor. Although the prognostic value of FST in various cancers has been investigated previously, studies rarely focused on hypopharyngeal carcinoma (HPC). In our study, the effect of FST expression on HPC tissues and cell lines was investigated., Methods: A total of 60 patients with HPC were recruited for this study. Levels of FST mRNA and protein were measured by quantitative polymerase chain reaction (PCR) and immunohistochemistry in HPC tissue samples and by qPCR in the HPC FaDu cells, as well as immortal nasopharyngeal epithelial cell line NP-69 cells. After silencing the FST expression in FaDu cells using lentivirus-mediated siRNA that was specific for FST mRNA, cell proliferation was determined by a Celigo assay. Tumor growth was monitored in nude mice and viability was determined by a methylthiazoletetrazolium assay. The ratio of cell cycle arrest and apoptosis was evaluated by flow cytometry. The colony formation ability was performed using Giemsa staining. In addition, wound healing and Transwell migration and invasion assays were performed for the analysis of cell motility., Results: FST expression was significantly higher in human HPC tissue and FaDu cells than in normal tissue and NP-69 cells. A higher expression of FST in HPC samples was positively correlated with advanced tumors. Moreover, FST knockdown by shRNA significantly decreased cell growth, colony formation, migration and invasion. Furthermore, FST silencing increased the cell apoptosis percentage, and arrested cell cycle in the S phase in FaDu cells. In addition, FST silencing suppressed tumor growth in vivo., Conclusions: Our results indicated that the FST gene was associated with HPC progression and may serve as a potential therapeutic target for the treatment of HPC., (© 2022. Huazhong University of Science and Technology.)

Background: Hypopharyngeal cancer is a relatively rare malignancy with poor prognosis. Current chemotherapeutic algorithm is still far from personalized medicine, and the identification of the truly active therapeutic biomarkers and/or targets is eagerly awaited., Methods: Venturing to focus on the conventional key chemotherapeutic drugs, we identified the most correlative genes (and/or proteins) with cellular sensitivity to docetaxel (TXT), cisplatin (CDDP) and 5-fluorouracil (5-FU) in the expression levels, through 3 steps approach: genome-wide screening, confirmation study on the quantified expression levels, and knock-down and transfection analyses of the candidates. The probable action pathways of selected genes were examined by Ingenuity Pathway Analysis using a large-scale database, The Cancer Genome Atlas., Results: The first genome-wide screening study derived 16 highly correlative genes with cellular drug sensitivity in 15 cell lines (|R| > 0.8, P < 0.01 for CDDP and 5-FU; |R| > 0.5, P < 0.05 for TXT). Among 10 genes the observed correlations were confirmed in the quantified gene expression levels, and finally knock-down and transfection analyses provided 4 molecules as the most potent predictive markers-AGR2 (anterior gradient 2 homolog gene), and PDE4D (phosphodiesterase 4D, cAMP-specific gene) for TXT; NINJ2 (nerve Injury-induced protein 2); CDC25B (cell division cycle 25 homolog B gene) for 5-FU- in both gene and protein expression levels. Overexpression of AGR2, PDE4D signified worse response to TXT, and the repressed expression sensitized TXT activity. Contrary to the findings, in the other 2 molecules, NINJ2 and CDC25, there observed opposite relationship to cellular drug response to the relevant drugs. IPA raised the potential that each selected molecule functionally interacts with main action pathway (and/or targets) of the relevant drug such as tubulin β chain genes for TXT, DNA replication pathway for CDDP, and DNA synthesis pathway and thymidylate synthetase gene for 5-FU., Conclusion: We newly propose 4 molecules -AGR2, PDE4D,NINJ2 and CDC25B) as the powerful exploratory markers for prediction of cellular response to 3 key chemotherapeutic drugs in hypopharyngeal cancers and also suggest their potentials to be the therapeutic targets, which could contribute to the development of precision medicine of the essential chemotherapy in hypopharyngeal patients. (339 words)., (© 2022. The Author(s).)

Objective: To evaluate whether the presence of human papillomavirus (HPV) DNA and p16 might be associated with better prognosis in patients with hypopharyngeal carcinoma (HPC), especially on overall survival (OS) and disease-free survival (DFS)., Method: PubMed, the Cochrane Library, the Web of Science and EMBASE were searched from inception to April 2021 to search for HPV DNA- and p16-related prognostic articles on HPC. Meta-analysis was performed on the selected articles according to the inclusion and exclusion criteria. Publication bias was assessed for the included studies with Egger's test. All studies were analyzed by using Stata 16.0 statistical software., Results: A total of 18 studies were included, including 12 HPV DNA studies and 11 p16 studies. Meta-analysis showed that HPV DNA positivity was a strong prognostic factor for improved OS in patients with HPC, with a pooled hazard ratio (HR) of 0.61 (95% CI, 0.54-0.69), but there was no statistically significant difference in DFS (HR, 0.60; 95% CI, 0.31-1.16). Patients with p16-positive tumors had better OS (HR, 0.66; 95% CI, 0.49-0.89) and DFS (HR, 0.59; 95% CI, 0.44-0.78) than patients with p16-negative tumors., Conclusions: This study suggests that the presence of HPV DNA leads to better OS in patients with HPC, and the presence of p16 also corresponds to better OS and DFS. Our results provide up-to-date evidence to clinicians and researchers. Larger studies adjusting for prognostic factors are needed in subsequent studies., (© 2022. The Author(s).)

Background: Lymphatic metastasis was an independent prognostic risk factor for hypopharyngeal carcinoma and was the main cause of treatment failure. The purpose of this study was to screen the differential genes and investigate the mechanism of lymphatic metastasis in hypopharyngeal carcinoma., Methods: Transcriptome sequencing was performed on primary tumors of patients, and differential genes were screened by bioinformatics analysis. The expression of differential genes was verified by qRT-PCR, western-blotting and immunohistochemical, and prognostic value was analyzed by Kaplan-Meier and log-rank test and Cox's test. Next, FADU and SCC15 cell lines were used to demonstrate the function of differential genes both in vitro by EdU, Flow cytometry, Wound Healing and Transwell assays and in vivo by a foot-pad xenograft mice model. Proteomic sequencing was performed to screen relevant targets. In addition, in vitro and in vivo experiments were conducted to verify the mechanism of lymphatic metastasis., Results: Results of transcriptome sequencing showed that RAF1 was a significantly differential gene in lymphatic metastasis and was an independent prognostic risk factor. In vitro experiments suggested that decreased expression of RAF1 could inhibit proliferation, migration and invasion of tumor cells and promote apoptosis. In vivo experiments indicated that RAF1 could promote tumor growth and lymphatic metastasis. Proteomic sequencing and subsequent experiments suggested that LAGE1 could promote development of tumor and lymphatic metastasis, and was regulated by RAF1., Conclusions: It suggests that RAF1 can promote lymphatic metastasis of hypopharyngeal carcinoma by regulating LAGE1, and provide a basis for the exploring of novel therapeutic target and ultimately provide new guidance for the establishment of intelligent diagnosis and precise treatment of hypopharyngeal carcinoma., (© 2022. The Author(s).)

Head and neck squamous cell carcinomas (HNSCC) are among the most common cancers worldwide and are associated with a poor prognosis for patients. Among HNSCC, those originating in the hypopharynx have the worst prognosis. The histone demethylase LSD1 has been shown to promote cancer initiation, progression, and relapse through various mechanisms and is upregulated in many cancer tissues. LSD1 physically interacts with SNAIL and is required for SNAIL mediated transcriptional repression. Previous studies of the prognostic value of LSD1 in HNSCC have been limited in their analysis of sub-sites, and a correlation between LSD1 and SNAIL has not been shown in HNSCC patient samples. Here we used a large, representative, and clinically well-characterized cohort of 339 HNSCC patients to investigate the co-expression of LSD1 and SNAIL and their prognostic value in all HNSCC using immunohistochemical staining. Elevated LSD1 expression correlated with advanced tumor stage and poor progression-free survival (PFS) in HNSCC originating in the hypopharynx. Overexpression of the transcription factor SNAIL independently correlated with worse overall survival (OS) and PFS in HNSCC in general and prominently in tumors of the hypopharynx. Furthermore, increased LSD1 expression significantly correlated with elevated SNAIL expression in patient samples. Therefore, the presented data implicates LSD1 and SNAIL as independent prognostic biomarkers.

Background: Hypopharyngeal squamous cell carcinoma (HSCC) is a common malignant cancer characterized by high metastasis and infiltration. The development of new approaches for the early diagnosis and identification of new therapeutic targets is essential. TIPE2 is well known as a tumor suppressor and related to a favorable prognosis of HSCC. However, its underlying mechanism remains unclear., Methods and Materials: TIPE2 expression was determined by immunohistochemistry and RT-qPCR. A TIPE2 overexpression stable cell line was generated by lentivirus infection. TIPE2 and other related protein levels were detected by western blotting. The cell cycle and apoptosis were performed by flow cytometric analysis. Cell proliferation was measured with a Cell Counting Kit-8 (CCK-8) assay, and the activity of caspase-3 and caspase-7 was assessed by Caspase-Glo® 3/7 Assay. All data were analyzed with SPSS 25 and GraphPad Prism 8.0., Results: TIPE2 expression was significantly down-regulated in HSCC. Low TIPE2 expression may be associated with poor prognosis in HSCC. TIPE2 overexpression markedly inhibited tumor cell migration. Moreover, TIPE2 decreased cell proliferation but promoted apoptosis. TIPE2 suppressed tumor growth by activating Epithelial-Mesenchymal Transition (EMT) and the extrinsic apoptosis pathway., Conclusion: TIPE2 inhibited tumor progression by suppressing cell migration but promoting apoptosis. TIPE2 can be a new therapeutic target in HSCC., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Programmed cell death-ligand 1 (PD-L1) have been attracting increasing attention in cancer diagnosis and treatment. The insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) is involved in the progression of multiple types of cancer. So, the role of IGF2BP2 and PD-L1 in hypopharyngeal carcinoma was assessed. Western blotting and immunochemistry were used to evaluate the expression of IGF2BP2 and PD-1/PD-L1. IGF2BP2 expression was knocked down in FaDu cells, and the effects on cell viability, apoptosis and proliferation were measured. A tumor-bearing nude model of hypopharyngeal carcinoma was constructed to evaluate the effect of a PD-L1 inhibitor and IGF2BP2 knockdown on hypopharyngeal carcinoma in vivo . RNA pull-down assays were used to assess the interaction between IGF2BP2 and PD-L1. The results showed that knockdown of IGF2BP2 inhibited FaDu cell proliferation and promoted apoptosis, as evidenced by the lower cell viability, a higher ratio of TUNEL-positive cells, decreased expression of Bcl-2 and cyclins, and increased expression of cleaved-caspase 3. In vivo , the tumor volume and weight were reduced by both the PD-L1 inhibitor and IGF2BP2 knockdown. Additionally, the interaction between PD-L1 and IGF2BP2 was confirmed. In conclusion, the results in the present study revealed that inhibition of IGF2BP2 might be a potentially relevant method for treating hypopharyngeal carcinoma, and the effects might be mediated via inhibition of the PD-1/PD-L1 axis.

Hypopharyngeal squamous cell carcinoma (HSCC) accounts 95% of hypopharyngeal cancer, which is characterized by high early metastasis rate and poor prognosis. It is reported that circular RNA is involved in the occurrence and development of cancer; however, the role of circRNA in hypopharyngeal cancer has little been investigated. We performed hypopharyngeal carcinoma circRNA microarray and qRT-PCR verification. The results showed circ&#95;0058106 expression level was significantly upregulated in tumor tissues than in corresponding normal tissues. We found that circ&#95;0058106 upregulation promoted proliferation, migration and invasion of HSCC cells, while knockdown of circ&#95;0058106 inhibited proliferation, migration and invasion of HSCC cells both in vitro and in vivo. Bioinformatics predicted circ&#95;0058106 may interact with miR-185-3p. We verified circ&#95;0058106 directly bound miR-185-3p and downregulated miR-185-3p expression by using dual-luciferase reporter assay and qRT-PCR. Moreover, we proved circ&#95;0058106 promoted HSCC cells tumorigenesis and EMT process by regulating Wnt2b/β-catenin/c-Myc pathway via miR-185-3p. In conclusion, our findings firstly confirmed the carcinogenic effect of circ&#95;0058106 in promoting HSCC cells tumorigenesis, metastasis, invasion and EMT process by regulating Wnt2b/β-catenin/c-Myc pathway through sponging miR-185-3p, indicating that circ&#95;0058106 may be a new therapeutic target and prognostic marker for HSCC., (© 2021. The Author(s).)

Laryngeal squamous cell carcinoma (LSCC) and hypopharyngeal squamous cell carcinoma (HSCC) seriously affect the life quality of patients. Nowadays, immunotherapy is widely used in the treatment of cancer. Tumor-infiltrating lymphocytes (TILs), programmed cell death 1 (PD-1) and its ligand programmed cell death ligand 1 (PD-L1) play key roles in the immunotherapy of cancer. Moreover, study has reported that the upregulation of PD-L1 and apurinic/apyrimidinic endonuclase 1 (APE1) are associated with tumorigenesis and poor prognosis of gastric cancer. In the present study, the number of CD3+ T lymphocytes and the expressions of PD-1 and PD-L1 in LSCC and HSCC were detected in clinical samples. In addition, the expressions of PD-L1 and APE1 and their correlation were explored. The results showed that PD-1+ T lymphocytes were wildly infiltrated and PD-L1 was overexpressed in LSCC and HSCC tissues. PD-1 had a positive correlation with cancer progression, and glottic and subglottic LSCC tissues might have a more active immune microenvironment. Moreover, the results showed that upregulated co-expression of PD-L1 and APE1 was a biomarker of LSCC, and APE1 could regulate the expression of PD-L1 through NF-κB signaling pathway. In conclusion, the combine detection of the expressions of PD-1, PD-L1 and APE1 will provide predictive value for the treatment of LSCC and HSCC via immune checkpoint inhibitors, which will help us to identify the patient population more likely to benefit from the immune checkpoint inhibitors based on the tumor immune microenvironment., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Actin-binding proteins (ABPs) and various signaling systems are involved in the process of squamous cell carcinoma of the larynx and hypopharynx (SCCLH) metastasis. The clinical significance of these proteins has not yet been determined. We analyzed the relationship between the mRNA levels of cofilin 1 (CFL1), profilin 1 (PFN1), adenylyl cyclase-associated protein 1 (CAP1), SNAI1 and RND3 and SCCLH metastasis. The serum levels of the above ABPs were estimated and the relationship between them and their mRNA expressions was analyzed. The expression levels of ABP mRNAs were measured by real-time RT-PCR in paired tissue samples taken from 54 patients with SCCLH (T1-4N0-1M0). Expression analysis was performed using the 2 -ΔΔCT method. The levels of ABPs in the blood serum were measured by ELISA. Statistical analysis was carried out using the SPSS Statistica 20.0 software package. No significant difference in the mRNA gene expression in tumor tissue of patients with T1-3N0M0 SCCLH and patients with T2-4N1-2M0 SCCLH was found. High expression of RND3 mRNA was accompanied by an increase in mRNA expression of all studied ABPs. In the blood serum of T2-4N1-2M0 patients, the level of PFN1 was lower by 21% and the level of CAP1 was higher by 75% than those observed in T1-4N0M0 patients. The data obtained showed that RND3 is involved in the regulation of molecular cascades of SCCLH metastasis. PFN1 and CAP1 serum levels can be good classifiers of metastases in patients with SCCLH., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Naturally occurring phytochemicals of different origin and structure, arctigenin, bergenin, usnic acid and xanthohumol, were shown to affect Nrf2 pathway in the context of various diseases, but their effect on this pathway in cancer cells was not extensively investigated. This study aimed to evaluate the effect of these compounds on Nrf2 expression and activation in hypopharyngeal FaDu squamous cell carcinoma cells. FaDu cells were treated with 2 or 10 μM arctigenin, bergenin, (+)-usnic acid or xanthohumol for 24 h. While arctigenin, bergenin, and xanthohumol did not affect either Nrf2 expression or activation, (+)-usnic acid treatment increased its transcript level and increased the nuclear/cytosol Nrf2 protein ratio-the measure of Nrf2 pathway activation. Consequently, (+)-usnic acid enhanced the transcription and translation of Nrf2 target genes: NQO1, SOD, and to a lesser extent, GSTP. The treatment of FaDu cells with (+)-usnic acid decreased both GSK-3β transcript and protein level, indicating its possible involvement in Nrf2 activation. All the tested compounds decreased Bax mRNA but did not change the level of Bax protein. (+)-Usnic acid tended to increase the percentage of early apoptotic cells and LC3 protein, autophagy marker. Significant induction of p53 also was observed after treatment with (+)-usnic acid. In summary, the results of this study indicate that low concentrations of (+)-usnic acid activate Nrf2 transcription factor, most probably as a result of ROS accumulation, but do not lead to FaDu hypopharyngeal carcinoma cells death.

Background: G protein-coupled receptor 12 (GPR12) is an orphan receptor with no confirmed endogenous ligands. It plays important roles in both physiological and pathological conditions such as neurogenesis and neural inflammation. However, it remains unclear whether GPR12 regulates carcinogenesis and progression in head and neck squamous cell carcinoma (HNSCC), such as esophageal cancer (EC) and hypopharyngeal cancer (HC)., Methods: The Cancer Genome Atlas (TCGA) database was applied to explore the expression of GPR12. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of GPR12 in cancer tissues. Wound healing and transwell assays were carried out to verify the effect of GPR12 on cell migration. Flow cytometric analysis and caspase-Glo 3/7 assay were carried out to verify the influence of GPR12 on cell apoptosis. Western blotting was used to measure the expression of proteins related to migration and apoptosis., Result: The qRT-PCR analyses showed that the expression of GPR12 decreased in EC and HC than that in their paired adjacent normal tissues. Wound healing assay and transwell assay demonstrated that GPR12 inhibited tumor cell migration. Flow cytometry analysis and Caspase-Glo 3/7 Assay suggested that GPR12 promoted apoptosis. The mechanism of GPR12 may function via modulating caspase-7, E-cadherin, and α-catenin in EC and HC cells., Conclusion: In conclusion, GPR12 induced apoptosis by activating caspase-7 and inhibited migration through epithelial-to-mesenchymal transition (EMT) in EC and HC. Our findings demonstrated that GPR12 as a potential tumor suppressor mediated cell migration and apoptosis in EC and HC., (© 2021 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.)

Hypopharyngeal squamous cell carcinoma (HPSCC) has a very poor prognosis. Local surgery may increase survival, but is often avoided due to significant post-op co-morbidities. Since prognostic markers are lacking, the aim was to find predictive biomarkers that identify patients whose response to oncological treatment is poor and who may benefit from primary surgery to increase survival. Pretreatment biopsies from 23 HPSCC patients, 3 human papillomavirus (HPV) positive and 20 HPV-negative, were analyzed for expression of 750 mRNAs using the Nanostring nCounter IO360 panel in relation to 3-year survival. Validation was performed through immunohistochemistry (IHC) for HLA class I and S100A12 in 74 HPV-negative HPSCC samples. Clustering identified a subset of HPV-negative HPSCC with favorable prognosis and a gene expression signature overexpressing calgranulins and immune genes, distinct from that of HPV-positive HPSCC. Enrichment analysis showed immune signaling, including the tumor inflammation signature, to be enriched in surviving patients. IHC validation confirmed high S100A12 and HLA class I expression to correlate with survival in HPV-negative HPSCC. This shows that immune activity is strongly related to survival in HPV-negative HPSCC. Enrichment of the tumor inflammation signature indicates a potential benefit of immunotherapy. Low expression of both HLA class I and S100A12 could be used to select patients for local surgery.

Objective: Hypopharyngeal squamous cell carcinoma (HSCC) is a common type of malignant tumor. Long non-coding RNAs (lncRNAs) are known to participate in HSCC development, while the role of lncRNA MALAT1 in HSCC remains largely unknown. We aimed to explore function of the lncRNA MALAT1/miR-429/ZEB1 axis in HSCC progression., Methods: Levels of MALAT1, miR-429 and ZEB1 in HSCC tissues samples were assessed. The FaDu cells were respectively treated with relative sequence or plasmid of MALAT1, miR-429, or ZEB1. Then, CCK-8 assay, colony formation assay, flow cytometry and Transwell assay were used to determine the cell proliferation, apoptosis, cell cycle, migration and invasion of the cells. The PI3K/Akt/mTOR signaling pathway-related proteins, proliferation-related proteins, cell cycle-related proteins, apoptosis-related proteins, and migration-related proteins were detected using Western blot analysis. The cell growth in vivo was observed. The targeting relationships between MALAT1 and miR-429, and between miR-429 and ZEB1 were confirmed., Results: MALAT1 and ZEB1 expression in HSCC was upregulated while miR-429 expression was downregulated. Reduced MALAT1 and ZEB1, and upregulated miR-429 inactivated the PI3K/Akt/mTOR signaling pathway, suppressed in vitro viability, colony formation ability, migration and invasion, as well as cell growth in vivo, and promoted the apoptosis of FaDu cells. Downregulated miR-429 reversed the role of MALAT1 inhibition in FaDu cell growth. LncRNA MALAT1 served as a sponge of miR-429, thus regulating ZEB1 expression., Conclusion: Inhibition of MALAT1 was able to elevate miR-429 to suppress the progression of HSCC via reducing ZEB1. Our research provided a potential therapeutic target for HSCC., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objective: To determine the tumor genomic, immunologic expression, and risk factors of treatment outcomes for patients with double head and neck squamous cell carcinoma (HNSCC) and esophageal squamous cell carcinoma (ESCC)., Methods: We reviewed patients with double HNSCC and ESCC between 1995 and 2014. The TP53 genomic mutation, CD8+ tumor infiltrating lymphocytes (TIL) and tumor programmed cell death ligand 1 (PD-L1) expression of paired HNSCC and ESCC were analyzed., Results: A total of 116 patients (57 metachronous and 59 synchronous) were included. There were 88 (75.86%) patients with HNSCC and 80 (68.97%) with ESCC harboured TP53 disruptive mutation. Nearly 106 (91.38%) patients had different clonality of TP53 mutation in paired HNSCC and ESCC. The immunologic expression of synchronous and metachronous patients was significantly different. Compared to the metachronous patients, the synchronous patients had significantly higher HNSCC CD8+ TIL (p = 0.03), ESCC CD8+ TIL (p < 0.001), HNSCC PD-L1+ tumor proportion score (TPS, p = 0.04), and ESCC PD-L1+ TPS (p = 0.04). Furthermore, among the synchronous patients, the immunologic expression between HNSCC and ESCC was significantly correlated. The CD8+ TIL and PD-L1 TPS had strongly (r = 0.63, p < 0.0001) and moderately (r = 0.42, p = 0.001) positive correlations, respectively. Finally, advanced stage (III/IV) HNSCC was a significant factor for disease-free (p = 0.03) and overall survival (p = 0.005)., Conclusion: In patients with double HNSCC and ESCC, nearly all HNSCC and ESCC were of multicentric origin. For the synchronous patients, there was more adaptive immune resistance in HNSCC and ESCC. The immunologic expression between paired HNSCC and ESCC was also significantly correlated., (Copyright © 2020. Published by Elsevier Ltd.)

The link between differences in molecular expression and survival among advanced laryngeal (LSCC) and hypopharyngeal squamous carcinoma (HPSCC) remains unclear. Here, we applied the Surveillance, Epidemiology, and End Results (SEER) program, Isobaric tag for relative and absolute quantitation (iTRAQ) with Liquid chromatography-mass spectrometry (LC-MS/MS) proteomics data and The Cancer Genome Atlas (TCGA) related data to discover the possible disparities between HPSCC and LSCC. Our results showed a significantly worse 5-year overall-survival in HPSCC compared with LSCC before and after adjusting for clinical parameters. 240 differentially expressed proteins were enriched in molecular networks of cytoskeleton remodeling and antigen presentation. Moreover, HPSCC consisted of less T-central-memory cells, T-follicular-helper cells, TGF-β response, and CD4  +   T memory resting cells, but more wound healing than LSCC. Furthermore, 9 mRNAs expression were  significantly and independently correlated to overall survival in 126 HPSCC and LSCC patients, which was further validated in another cohort of head and neck cancers. These findings support that Immunity signatures as well as pathway networks that include cytoskeleton remodeling and antigen presentation may contribute to the observed differences in survival between HPSCC and LSCC.

Emerging studies have demonstrated that long non‑coding RNAs (lncRNAs) play essential roles in tumorigenesis. However, the role and function of lncRNAs in hypopharyngeal squamous cell carcinoma (HSCC) have not been completely elucidated. The present study explored the function of a novel lncRNA, RP11‑156L14.1, in HSCC. RP11‑156L14.1 was revealed to be highly expressed in HSCC tissues and cell lines. Knockdown of RP11‑156L14.1 inhibited proliferation, migration, and invasion in HSCC cells. Furthermore, RP11‑156L14.1 regulated epithelial‑mesenchymal transition (EMT) by controlling EMT‑related protein expression. Mechanistically, RP11‑156L14.1 exerted its function as a competing endogenous RNA (ceRNA) and directly interacted with miR‑548ao‑3p. The present study also demonstrated that miR‑548ao‑3p regulated signal sequence receptor subunit 1 (SSR1) expression by targeting SSR1 3'‑UTR. Moreover, the xenograft HSCC tumor model revealed that knockdown of RP11‑156L14.1 markedly suppressed HSCC tumor growth in vivo. In summary, these findings indicated that the lncRNA RP11‑156L14.1 functions as an oncogene in HSCC by competing with miR‑548ao‑3p in regulating SSR1 expression. The RP11‑156L14.1/miR‑548ao‑3p/SSR1 axis could be utilized as a potential novel biomarker and therapeutic target for HSCC.

Background: Among head and neck squamous cell carcinomas (HNSCCs), hypopharyngeal squamous cell carcinoma (HPSCC) has the worst prognosis. Iron metabolism, which plays a crucial role in tumor progression, is mainly regulated by alterations to genes and post-transcriptional processes. The recent discovery of the N6-methyladenosine (m 6 A) modification has expanded the realm of previously undiscovered post-transcriptional gene regulation mechanisms in eukaryotes. Many studies have demonstrated that m 6 A methylation represents a distinct layer of epigenetic deregulation in carcinogenesis and tumor proliferation. However, the status of m 6 A modification and iron metabolism in HPSCC remains unknown. Methods: Bioinformatics analysis, sample analysis, and transcriptome sequencing were performed to evaluate the correlation between m 6 A modification and iron metabolism. Iron metabolic and cell biological analyses were conducted to evaluate the effect of the m 6 A reader YTHDF1 on HPSCC proliferation and iron metabolism. Transcriptome-wide m 6 A-seq and RIP-seq data were mapped to explore the molecular mechanism of YTHDF1 function in HPSCC. Results: YTHDF1 was found to be closely associated with ferritin levels and intratumoral iron concentrations in HPSCC patients at Sir Run Run Shaw Hospital. YTHDF1 induced-HPSCC tumorigenesis depends on iron metabolism in vivo in vitro . Mechanistically, YTHDF1 methyltransferase domain interacts with the 3'UTR and 5'UTR of TRFC mRNA, then further positively regulates translation of m 6 A-modified TFRC mRNA. Gain-of-function and loss-of-function analyses validated the finding showing that TFRC is a crucial target gene for YTHDF1-mediated increases in iron metabolism. Conclusion: YTHDF1 enhanced TFRC expression in HPSCC through an m 6 A-dependent mechanism. From a therapeutic perspective, targeting YTHDF1 and TFRC-mediated iron metabolism may be a promising strategy for HPSCC., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Studies have confirmed that microRNAs play important roles in the development and progression of cancer. Therefore, to identify the differentially expressed microRNAs between the cancer and the normal tissues, microRNAs will provide new clues for exploring the molecular mechanisms of cancer development and potential targeted therapies. In the present study, we found that miR-338-3p was downregulated in hypopharyngeal carcinoma and inversely correlated with the pathological grade. When the miR-338-3p was further downregulated, the migration and invasion ability of the FaDu hypopharyngeal carcinoma cells were enhanced, and these functions were inhibited when the miR-338-3p was upregulated. In addition, we demonstrated that ADAM17 was a target of miR-338-3p, and that ADAM17 directly activated the wnt/β-catenin signaling pathway and promoted the expression of its target gene MMP2, Nanog and SOX2, which affected the growth, migration and invasion of hypopharyngeal carcinoma cells. In conclusion, our results demonstrate for the first time that miR-338-3p targets ADAM17 and blocks the development of hypopharyngeal carcinoma involving the wnt/β-catenin signaling pathway, which may be a new target for clinical intervention in human cancer.

Hypopharyngeal squamous cell carcinoma (HSCC) is a malignant tumor found in the head and neck region. Lactate receptor 1, also known as G protein‑coupled receptor81 (GPR81), has been reported to play a vital role in cancer growth and metabolism. However, the function of GPR81 in HSCC remains largely unknown. The present study investigated the effect of GPR81 on cell survival and GPR81‑mediated energy metabolism under cisplatin treatment in HSCC. GPR81 knockdown reduced the proliferation and invasion of the human HSCC cell line FaDu. Furthermore, GPR81 silencing combined with cisplatin treatment increased the expression of translocase of outer mitochondrial membrane 20 at the mRNA and protein levels (P<0.05). mRNA and protein expression of phosphofructokinase 1 in mRNA appeared to be downregulated in GPR81 knockdown FaDu cells treated with cisplatin, although this was not statistically significant. GPR81 silencing and cisplatin challenge showed no significant upregulation compared with the control, but significant downregulation in mRNA and protein levels compared with the shRNA‑scramble group. Apoptosis was measured by flow cytometry with annexin V and 7‑aminoactinomycin D. GPR81 silencing and cisplatin led to an increased apoptotic rate. Moreover, absence of GPR81 combined with cisplatin exposure increased caspase‑3 expression and decreased Bcl‑2 levels. The results of the present study suggested that GPR81 and cisplatin sensitivity played an important role in HSCC growth and metabolism.

Hypopharyngeal cancer (HPC) accounts for the lowest survival rate among all types of head and neck cancers (HNSCC). However, the therapeutic approach for HPC still needs to be investigated. In this study, a theranostic 188 Re-liposome was prepared to treat orthotopic HPC tumors and analyze the deregulated microRNA expressive profiles. The therapeutic efficacy of 188 Re-liposome on HPC tumors was evaluated using bioluminescent imaging followed by next generation sequencing (NGS) analysis, in order to address the deregulated microRNAs and associated signaling pathways. The differentially expressed microRNAs were also confirmed using clinical HNSCC samples and clinical information from The Cancer Genome Atlas (TCGA) database. Repeated doses of 188 Re-liposome were administrated to tumor-bearing mice, and the tumor growth was apparently suppressed after treatment. For NGS analysis, 13 and 9 microRNAs were respectively up-regulated and down-regulated when the cutoffs of fold change were set to 5. Additionally, miR-206-3p and miR-142-5p represented the highest fold of up-regulation and down-regulation by 188 Re-liposome, respectively. According to Differentially Expressed MiRNAs in human Cancers (dbDEMC) analysis, most of 188 Re-liposome up-regulated microRNAs were categorized as tumor suppressors, while down-regulated microRNAs were oncogenic. The KEGG pathway analysis showed that cancer-related pathways and olfactory and taste transduction accounted for the top pathways affected by 188 Re-liposome. 188 Re-liposome down-regulated microRNAs, including miR-143, miR-6723, miR-944, and miR-136 were associated with lower survival rates at a high expressive level. 188 Re-liposome could suppress the HPC tumors in vivo, and the therapeutic efficacy was associated with the deregulation of microRNAs that could be considered as a prognostic factor.

BACKGROUND The ubiquitin-proteasome pathway (UPP) is closely associated with the occurrence and progression of cancer, and the 5i immunoproteasome subunit is an important antitumor target in UPP. This study aimed to characterize the regulation of the immunoproteasome subunit ß5i (PSMB8) in JHU-011 laryngeal carcinoma cells and FaDu hypopharyngeal carcinoma cells to explore a new target for the treatment of laryngeal and hypopharyngeal carcinomas. MATERIAL AND METHODS JHU-011 and FaDu cells were used as effector cells in this study. By means of ⁶⁰Co γ-irradiation, the construction of stable cell lines of the silenced proto-oncogene c-Abl, and the addition of exogenous tyrosine kinase inhibitor (TKI) and activator, the transcription and protein expression levels of PSMB8 and its alternatively spliced isoforms in both cell lines were detected by real-time fluorescence quantitative polymerase chain reaction (RT-PCR) and Western blot. RESULTS Ionizing radiation upregulated the transcription level of the alternatively spliced isoform of PSMB8, E2, in both cell lines, thereby upregulating the mRNA and protein levels of PSMB8. The silencing of the proto-oncogene c-Abl and the activation and inhibition of its kinetic kinase product can affect the transcription and protein levels of PSMB8. CONCLUSIONS Ionizing radiation can significantly upregulate the mRNA and protein levels of PSMB8, which happens through the upregulation of its splicing isoform E2. The proto-oncogene c-Abl and its kinetic kinase protein product can regulate the transcription and protein expression levels of PSMB8 and its alternatively spliced isoforms.

Background: Prognosis for hypopharyngeal cancer is usually poor, and recurrence is common. Identifying new factors or related mechanisms that promote its progression may have clinical implications. Although, recent studies support bile reflux in hypopharyngeal carcinogenesis, it remains to be explored how bile and its related NF-κB activated pathway may further affects its progression in already established hypopharyngeal cancer., Methods: Hypopharyngeal squamous cell carcinoma (HSCC) cell lines, FaDu and UMSCC11A, both negative for HPV, were repetitively exposed to bile acids (400 μM) at variable pH points (4.0, 5.5 and 7.0). Immunofluorescence, western blotting, luciferase assay, and qPCR were used to detect NF-κB activation, bcl-2 overexpression and gene expression., Results: Bile at strongly acidic pH (4.0) potentiated the activation of NF-κB and its related mRNA phenotype in HSCC cells. IL-6, TNF-α, and BCL2 were found among the highest overexpressed genes as was previously found in HSCCs excised from patients with documented biliary reflux. An enhanced transcriptional activity of EGFR, RELA, STAT3, and WNT5Α and higher survival rates were observed in HSCC cells exposed to acidic bile compared to those exposed to bile at weakly acidic or neutral pH., Conclusion: Our novel findings support the observation that bile reflux has the potential for actively influencing the progression of hypopharyngeal cancer, mediated by NF-κB. In patients with hypopharyngeal cancer and known gastroesophageal reflux disease, antacid therapy may exert a role in furthering control of disease recurrence and progression., Competing Interests: Declaration of Competing Interest The authors whose names are listed in this article certify that they have NO affiliations with or involvement in any organization or entity with any financial interest, or non-financial interest in the subject matter or materials discussed in this manuscript., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Hypopharyngeal squamous cell carcinoma (HSCC) remains one of the most lethal malignancies in the head and neck. Long noncoding RNA (lncRNA) HOXA11-AS is proven to function as an oncogene and a therapeutic target in various tumors. Our previous study and others have demonstrated that HOXA11-AS is one of the most upregulated lncRNAs in HSCC. However, the role of HOXA11-AS in HSCC has not yet been identified. The current study demonstrated that the expression of HOXA11-AS was significantly upregulated in HSCC tumors and was positively associated with lymph node metastasis. Moreover, functional experiments revealed that HOXA11-AS knockdown suppressed the proliferation and migration potential in FaDu cells. Furthermore, luciferase reporter gene assay combined with cellular functional experiments demonstrated that HOXA11-AS functioned as a molecular sponge for miR-155, and inhibition of miR-155 attenuated the suppressive effect of HOXA11-AS knockdown on the aggressive phenotype in HSCC. This study identifies a tumor-promoting role of HOXA11-AS in HSCC and suggests HOXA11-AS might be a potential diagnostic and therapeutic target for HSCC.

The purpose of this study was to identify potential miRNAs and mRNAs involved in chemotherapy insensitivity in hypopharyngeal squamous cell carcinoma (HSCC) and to explore the underlying mechanisms involved to provide diagnostic markers and therapeutic targets for HSCC. We used microarrays to identify differences in both the mRNA and miRNA expression profiles between a group (twelve patients) sensitive to chemotherapy and a resistant group (nine patients). We then employed bioinformatics tools to examine the functions and pathways involved. The genes and miRNAs most related to chemotherapy sensitivity in HSCC were screened. Finally, a miRNA-mRNA-phenotype network was constructed with an integrated analysis based on the identified miRNAs and mRNAs. Nine differentially expressed miRNAs and one hundred differentially expressed mRNAs were identified, and the functions of these genes and miRNAs were predicted. Bioinformatics analysis revealed a regulatory network consisting of eight genes and two miRNAs that influenced HSCC chemosensitivity. According to our analysis, CCL4L1 may be a potential molecular marker for HSCC chemotherapy, and excess CCL4L1 leads to the upregulation of PRAME and the downregulation of miR-375, thus decreasing HSPB8 expression and promoting chemotherapy sensitivity. Our work provides reliable data for further studies investigating the mechanism of HSCC chemotherapy sensitivity.

The present study aimed to investigate the clinical relevance of circulating tumor cells (CTCs) in patients with locally advanced head and neck squamous cell carcinoma (LA‑HNSCC), particularly in patients with nasopharyngeal and hypopharyngeal squamous cell carcinoma. CTCs were isolated using negative immunomagnetic bead enrichment and were identified by fluorescence in situ hybridization. Youden's index and the receiver operating characteristic (ROC) curve were used to select the optimal CTC baseline value. χ2 test or Fisher's test were used to determine the association between CTC counts and clinical parameters, curative effects and prognosis. The Kaplan‑Meier estimator was used to analyze overall survival (OS) and progression‑free survival (PFS). In the present study, 356 peripheral blood samples (178 pretreatment samples and 178 post‑treatment samples) from 178 patients were examined. The results revealed that the pretreatment CTC detection rate was 73.8%. The minimum, maximum and median CTC counts were 1, 22 and 2/3.2 ml, respectively. The number of polyploid CTCs was associated with distant metastasis (P=0.026). In addition, patients with undetectable CTCs, and decreasing or negative CTCs post‑treatment tended to have a good prognosis (P<0.05). For nasopharyngeal squamous cell carcinoma, the PFS of patients with increased CTCs and CTCs ≥2/3.2 ml after treatment was significantly lower (P<0.05). For hypopharyngeal squamous cell carcinoma, it was suggested that CTCs with a cutoff value of 3 may be used to evaluate PFS and OS before and after treatment. In conclusion, CTCs may be used to monitor disease progression and the response to chemoradiotherapy for patients with LA‑HNSCC. Furthermore, CTCs are a better predictor of the prognosis of hypopharyngeal squamous cell carcinoma than that of nasopharyngeal squamous cell carcinoma.

Background: The gene encoding fibroblast growth factor receptor 1 (FGFR1) is emerging as a therapeutic and prognostic biomarker in various cancer types, including head and neck squamous cell carcinoma (SCC). Here, we investigated the clinicopathologic implication of FGFR1 gene amplification and protein overexpression in hypopharyngeal and laryngeal SCC., Methods: Fluorescence in situ hybridization and immunohistochemistry were performed to determine FGFR1 gene amplification and protein overexpression in 209 surgically resected cases., Results: FGFR1 amplification observed in 8 (8/66, 12.1%; 6 hypopharynx and 2 larynx) patients and high FGFR1 expression in 21 (21/199, 10.6%) patients significantly correlated with lymph node metastasis and advanced pathological stages. FGFR1 amplification was also associated with worse disease-free survival in multivariate analysis (hazard ratio = 4.527, P = 0.032). High FGFR1 expression was more frequently observed, consistent with the worsening of the degree of histologic differentiation., Conclusions: FGFR1 amplification may serve as an independent prognostic factor for disease-free survival in hypopharyngeal and laryngeal SCC. Aberrant FGFR signaling caused by FGFR1 gene amplification or protein overexpression may play a crucial role in the malignant evolution and progression of hypopharyngeal and laryngeal SCC, and offer novel therapeutic opportunities in patients with hypopharyngeal and laryngeal SCC that usually lack specific therapeutic targets.

Objective: This work focused on the function role and underlying mechanism of BLACAT1 in regulating the radiosensitivity of head and neck squamous cell carcinoma (HNSCC) cells via PSEN1., Methods: BLACAT1 and PSEN1 expression in HNSCC tissues and cells were measured by qRT-PCR. Kaplan-Meier method and Spearman's correlation analysis determined the prognostic roles and association of BLCAT1 and PSEN1 in HNSCC. The impacts of BLACAT1 and PSEN1, alone and in combination, on radiosensitivity of HNSCC cells were separately assessed through CCK-8, colony formation, flow cytometry, western blot and γH2AX foci staining assays., Results: Our study disclosed that BLACAT1 and PSEN1 were both in association with poor prognosis and radioresistance of HNSCC cells. BLACAT1 knockdown improved the radiosensitivity of HNSCC cells by changing cellular activities containing repressed cell viability, accelerated cell apoptosis, induced cell cycle arrest, and stimulated DNA damage response. Further, we found that PSEN1 was positively correlated with BLACAT1. Rescue assays confirmed that BLACAT1 regulated the radiosensitivity of HNSCC cells by modulating PSEN1., Conclusion: We revealed that BLACAT1 knockdown enhanced radioresistance of HNSCC cells via regulating PSEN1, exposing the probable target role of BLACAT1 in HNSCC., Advances in Knowledge: This was the first time that the pivotal role of BLACAT1 was investigated in HNSCC, which provided a novel therapeutic direction for HNSCC patients.

Objective: To analyze the differentially expressed genes related to the chemosensitivity with the TPF regimen for hypopharyngeal squamous cell carcinoma and to measure potential functional targeting genes expressions. Methods: Twenty-nine patients with primary hypopharyngeal cancer who underwent induction chemotherapy with TPF from January 2013 to December 2017 in Beijing Tongren Hospital were enrolled for microarray analysis, including 28 males and 1 female, aged from 43 to 73 years old. Among them, 16 patients were sensitive to chemotherapy while 13 patients were non-sensitive. Illumina Human HT-12 Bead Chip was applied to analyze the gene expressions and online bioinformatics analysis was used to analyze the differentially expressed genes. Reverse transcription and quantitative real-time PCR (RT-qPCR) was used to measure the mRNA expression of potential functional genes of TPF induction chemotherapy in 43 samples, 29 from original patients and 14 from additional patients. Graphpad prism 7.0 software was used for statistical analysis. Results: A total of 1 381 significantly differentially expressed genes were screened out. By GO analysis, up-regulated genes included sequestering in extracellular matrix, chemokine receptor binding and potassium channel regulator activity; down-regulated genes included regulation of angiogenesis, calcium ion binding and natural killer cell activation involved in immune response. With KEGG database analysis, down-regulated pathways included ECM-receptor interaction and peroxisome and up-regulated pathways included Glutathione metabolism and PPAR signaling pathway. The expressions of CD44 and IL-6R were significantly different and appeared biologically significant. CD44 was significantly upregulated in insensitive tissues (0.54±0.06) compared with sensitive tissues (0.33±0.04)( P< 0.01). IL-6R was significantly downregulated in insensitive tissues (0.44±0.03) compared with sensitive tissues. (0.68±0.03) ( P< 0.01). Conclusion: CD44 and IL-6R may be potentially functional genes of TPF induction chemotherapy in hypopharyngeal squamous cell carcinoma.