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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).)

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 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).)

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).)

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.

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).)

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.

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.

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).)

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.

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.

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.

Background: Increased research efforts have demonstrated that lncRNAs are associated with multiple head and neck tumors and play important roles in cancer. We previously found that RP11-169D4.1-001 plays a tumor-suppressive role in laryngeal cancer, but its function in human hypopharyngeal squamous cell carcinoma (HSCC) remains unknown. Thus, this research aimed to analyze the relationship between RP11-169D4.1-001 expression and HSCC clinicopathological features., Methods: Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect the expression of RP11-169D4.1-001 in 70 pairs of HSCC and adjacent normal tissues., Results: The expression level of RP11-169D4.1-001 in HSCC tissues was significantly lower than that in adjacent normal tissues (P = .001). The expression of RP11-169D4.1-001 had no significant relationship with tumor differentiation, stage, smoking, drinking, age, tumor location, or treatment. RP11-169D4.1-001 expression was associated with T category (P = .008) and lymph node metastasis (P = .001). Survival data were assessed by Kaplan-Meier curves. Patients with high RP11-169D4.1-001 expression were found to have a shorter overall survival than patients with low RP11-169D4.1-001 expression. Multivariate analysis also indicated that target RNA was an independent factor for prognosis. The ROC curve was constructed to clarify the diagnostic value of RP11-169D4.1-001., Conclusions: RP11-169D4.1-001 may serve as a new biomarker and potential drug target and can be used as a new biomarker and a potential drug target for the detection and treatment of hypopharyngeal cancer, respectively. Furthermore, RP11-169D4.1-001 expression may be an independent prognostic factor affecting the survival of hypopharyngeal cancer patients., (© 2019 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals, Inc.)

Purpose: The BTF3 is involved in oncogenesis, while the biological roles in HSCC remain unclear. The aim of this study was to explore the impact of BTF3 knockdown on biological phenotypes of human HSCC in vivo and in vitro., Methods: The expression of BTF3 was assessed in HSCC and normal tissues. In vitro experiments were performed to explore impact of BTF3 knockdown on biological phenotypes of human HSCC cell line, including proliferation, cell cycle, and apoptosis. Moreover, nude mice were used to evaluate growth of xenograft tumors. Finally, gene chip was used to explore the potential signaling pathways of BTF3, with confirmation of potential BTF3-related genes., Results: Our results showed elevated expression of BTF3 was observed in HSCC tumors compared to paired adjacent normal tissues in 68 patients, and positively associated with lymph node metastasis and survival of this HSCC patient cohort. In addition, in vitro experiments showed that BTF3 knockdown significantly impaired regulation of proliferation, cell cycle, and apoptosis, potentially via ATM signaling pathway. Finally, in vivo experiments demonstrated that BTF3 functioned as an oncogene by promoting the development and progression of HSCC tumors, indicating its oncogenic role in HSCC., Conclusions: This study for the first time demonstrated that expression of BTF3 is upregulated in HSCC tumors and this upregulation is positively correlated with lymph node metastasis of this malignancy. The oncogenic role of BTF3 is further validated for tumor promotion and progression of HSCC in vivo, indicating that BTF3 is a potential therapeutic target and prognostic biomarker for HSCC., (Copyright © 2019 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Napabucasin is an NAD(P)H:quinone oxidoreductase 1 (NQO1)-bioactivatable small molecule hypothesized to affect multiple oncogenic pathways. In a prespecified, retrospective analysis of the napabucasin phase III CO.23 study, overall survival was longer for napabucasin versus placebo in patients expressing phosphorylated STAT3 (pSTAT3) in tumor cells and cells of the tumor microenvironment (TME). We hypothesized that a connection may exist between NQO1 expression in cancer cells and pSTAT3 in tumor cells and the TME. In 3D spheroid cocultures of cancer cells and cancer-associated fibroblasts, the antitumor activity of napabucasin was NQO1 dependent. The levels of cytokines such as IL6, CXCL10, and GM-CSF were higher in NQO1-positive versus NQO1-deleted cocultures. These differentially secreted cytokines promoted STAT3 phosphorylation in tumor cells and the TME. NQO1-expressing, napabucasin-sensitive tumor cells can modify tumor cells and the TME to promote STAT3 phosphorylation, suggesting that pSTAT3 may be used to identify a subpopulation of patients who would likely respond to napabucasin. IMPLICATIONS: pSTAT3 is a potential biomarker for patient response to the anticancer drug napabucasin. Visual Overview: http://mcr.aacrjournals.org/content/molcanres/17/7/1429/F1.large.jpg., (©2019 American Association for Cancer Research.)

Non-coding genetic variants play an important role in driving susceptibility to complex diseases but their characterization remains challenging. Here, we employed a novel approach to interrogate the genetic risk of such polymorphisms in a more systematic way by targeting specific regulatory regions relevant for the phenotype studied. We applied this method to meningococcal disease susceptibility, using the DNA binding pattern of RELA - a NF-kB subunit, master regulator of the response to infection - under bacterial stimuli in nasopharyngeal epithelial cells. We designed a custom panel to cover these RELA binding sites and used it for targeted sequencing in cases and controls. Variant calling and association analysis were performed followed by validation of candidate polymorphisms by genotyping in three independent cohorts. We identified two new polymorphisms, rs4823231 and rs11913168, showing signs of association with meningococcal disease susceptibility. In addition, using our genomic data as well as publicly available resources, we found evidences for these SNPs to have potential regulatory effects on ATXN10 and LIF genes respectively. The variants and related candidate genes are relevant for infectious diseases and may have important contribution for meningococcal disease pathology. Finally, we described a novel genetic association approach that could be applied to other phenotypes.

The in vitro efficacy of cancer prodrugs varies significantly between malignant cell lines. The most commonly identified problems relate to delivery: uptake mechanism, endosomal entrapment, and drug release. Here we present the study of collagen/cell penetrating hybrid (COL/CPP) peptide carriers intended to deliver paclitaxel to the hypopharyngeal carcinoma (FaDu) cells. Confocal microscopy imaging revealed the surprising response of FaDu cell to COL/CPP in comparison to previously studied cancer cell lines: hybrid peptides that carry both COL and CPP domain adsorb on the FaDu cell surface. While the CPP domain was design to facilitate the cellular uptake, in the case of FaDu cells, it also induced detrimental interactions with the cell membrane. Despite surface adsorption, the colocalization study with endosomal markers EEA1 and LAMP1 reveals that COL/CPP is internalized via endosomal pathway, peptides are able to escape before lysosome formation and release paclitaxel. Therefore, the main obstacle for paclitaxel delivery to FaDu cells appears to be related to cell surface properties. This behavior seems specific to FaDu cells, and could be linked to previously reported overexpression of T5, heparanase splice variants that produces protein lacking enzymatic activity of heparanase. This results in increased concentration of HSPG on FaDu cell surface, and possibly creates a barrier for cellular uptake of highly charged COL/CPP.

Accumulating evidence has suggested that circular RNAs (circRNAs), a novel class of non‑coding RNAs, have crucial roles in tumor progression. However, the significance of circRNAs in hypopharyngeal cancer (HCa) remains to be investigated. The present study has identified aberrantly expressed circRNAs by performing circRNA sequencing analyses of three pairs of tumor and adjacent normal samples from patients with HCa. The results demonstrated that 173 circRNAs were differentially expressed (DE), including 71 upregulated and 102 downregulated circRNAs (FDR<0.05 and fold changes of ≥2 or ≤0.5 by Mann‑Whitney U test followed by Benjamini‑Hochberg correction for multiple testing). Pathway analyses of the genes producing DE circRNAs revealed that many of them were involved in cancer‑related pathways. To further illustrate the roles of circRNAs in HCa progression, a competing endogenous RNA (ceRNAs) network was constructed, consisting of circRNAs, miRNA, and miRNA targeted genes. The results demonstrated that multiple cancer‑related pathways were affected by performing enrichment analyses of the targeted genes. Of note, a ceRNA subnetwork was isolated, consisting of two circRNAs (hsa&#95;circ&#95;0008287 and hsa&#95;circ&#95;0005027) and one miRNA (hsa‑miR‑548c‑3p), which significantly affect both ErbB and Hippo signaling pathways. In conclusion, the present study identified a set of circRNAs that are potentially implicated in the tumorigenesis of HCa and may serve as potential biomarkers for the diagnosis of HCa.

Hypopharyngeal carcinoma (HC) is one of the most malignant tumors in the upper aerodigestive tract. Currently, there are no effective treatments for HC. Gold nanoparticles (AuNPs) are a promising tool that can be used for plasmonic photothermal therapy (PPTT), which refers to the use of electromagnetic radiation, most often in near infrared (NIR) region, for the treatment of various medical conditions including cancer. AuNPs have been proved to be a promising tool for NIR spectroscopy-mediated photothermal therapies. In this study, we chemically conjugated AuNPs with a monoclonal antibody (mAb) targeting the epidermal growth factor receptor (EGFR), a cell-surface receptor that is overexpressed in many cancers. We then assessed the effect of NIR photothermal treatment with the EGFRmAb-AuNPs in FaDu HC cells. Our data showed that nanoparticle conjugation with the EGFRmAb improved the specific targeting towards FaDu cells and reduced cytotoxicity towards normal (293 T) cells which do not overexpress the EGFR. A significant amount of our EGFRmAb-conjugated AuNPs could enter the nucleus. Moreover, the expression levels of double strand DNA break repair proteins, including p-ATR, p-CHK1, and p-CHK2 were increased following AuNPs treatment, indicating the presence of DNA damage. These findings suggest that the AuNPs can potentially disrupt genome integrity and induce apoptosis. In addition, EGFRmAb-AuNPs+NIR could induce FaDu cell apoptosis, accompanied by the inhibition of the PI3K/AKT/mTOR pathway and stimulation of DNA damage response. Based on these data, PPTT using the EGFRmAb-AuNPs could be a new promising treatment for HC.

Treatment with analogues of the SERCA-inhibitor Thapsigargin is a promising new approach for a wide variety of cancer entities. However, our previous studies on various tumor cells suggested resistance of SEC62 over-expressing tumors to this treatment. Therefore, we proposed the novel concept that e.g. lung-, prostate-, and thyroid-cancer patients should be tested for SEC62 over-expression, and developed a novel therapeutic strategy for a combinatorial treatment of SEC62 over-expressing tumors. The latter was based on the observations that treatment of SEC62 over-expressing tumor cells with SEC62-targeting siRNAs showed less resistance to Thapsigargin as well as a reduction in migratory potential and that the siRNA effects can be mimicked by the Calmodulin antagonist Trifluoperazine. Therefore, the combinatorial treatment of SEC62 over-expressing tumors was proposed to involve Thapsigargin and Trifluoperazine. Here, we addressed the impact of Thapsigargin and Trifluoperazine in separate and combined treatments of heterotopic tumors, induced by inoculation of human hypopharyngeal squamous cell carcinoma (FaDu)-cells into the mouse flank. Seeding of the tumor cells and/or their growth rate were significantly reduced by all three treatments, suggesting Trifluoperazine is a small molecule to be considered for future therapeutic strategies for patients, suffering from Sec62-overproducing tumors.

BACKGROUND Ras-related C3 botulinum toxin substrate 1 (Rac1) is implicated in a variety of cellular functions and is related to tumor growth and metastasis. This study aimed to explore the role of Rac1 in hypopharyngeal squamous cell carcinoma (HSCC). MATERIAL AND METHODS The Rac1 expression in HSCC tissues was determined by quantitative real-time polymerase chain reaction and Western blot analysis. The level of Rac1 in HSCC cells was downregulated by a Rac1-specific shRNA. Then, the growth and metastasis of HSCC cells were assessed in vitro by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay, flow cytometry, Hoechst staining, and Transwell assay. Moreover, cells transfected with Rac1 shRNA or negative control were injected subcutaneously into the right axilla of mice, and then the effects of Rac1 silencing on the growth of HSCC were also explored in vivo. Additionally, activation of the P38 mitogen-activated protein kinase (MAPK) signaling pathway was assessed by Western blot. RESULTS Rac1 was highly expressed in HSCC tissues. Silencing Rac1 inhibited the proliferation and cell cycle progress of HSCC cells, and induced their apoptosis. Rac1 silencing also suppressed the migration and invasion of HSCC cells. In vivo study showed that silencing Rac1 suppressed the growth of tumor bodies. Moreover, the P38 MAPK signaling pathway was implicated in the tumor-suppressing effect of Rac1 silencing in vitro and in vivo. CONCLUSIONS Silencing Rac1 suppressed the growth and migration of HSCC through the P38 MAPK signaling pathway. Due to its contribution in HSCC, Rac1 has the potential to become a promising antitumor therapeutic target for HSCC.

Background: TP53 is the most frequently mutated gene in human cancers. Previous studies reported that TP53 mutations correlated with poor prognoses in patients with head and neck squamous cell carcinoma (HNSCC). However, the relationship between TP53 mutations and hypopharyngeal squamous cell carcinoma (HPSCC) is not known. The current study aimed to evaluate TP53 mutation status as a predictive biomarker in patients with HPSCC., Methods: We retrospectively reviewed the clinical charts of 57 HPSCC patients treated with initial surgery between 2008 and 2014. TP53 mutation status was determined by Sanger sequencing, and patients were classified into wild-type, missense mutation, and truncating mutation groups. Additionally, p53 expression was determined using immunohistochemistry in surgical specimens., Results: TP53 mutations were identified in 39 (68%) patients. The 3-year disease-specific survival (DSS) rate of wild-type, missense mutation, and truncating mutation group were 94%, 61%, and 43%, respectively. The TP53 mutation group displayed significantly worse DSS and overall survival rates than the wild-type group (P = 0.01 and P = 0.007, respectively). Multivariate analyses revealed that the presence of TP53 mutations and ≥4 metastatic lymph nodes were independent adverse prognostic factors for HPSCC. p53 immunopositivity was detected in 22 patients, including 5 (28%) and 17 (71%) patients in the wild-type and missense mutation groups, whereas none of the patients with truncating mutation exhibited p53 immunopositivity (P = 0.0001)., Conclusion: The TP53 mutation status correlated with poor prognosis in surgically treated HPSCC patients. Specifically, truncating mutations which were not detected by p53 immunohistochemistry were predictive of worst survival.

Colchicine is an alkaloid widely used for the treatment of inflammatory diseases, such as gout. It suppresses cell division by inhibiting mitosis. We investigated the anticancer effects of colchicine on human hypopharyngeal cancer cells and the mechanisms underlying its anticancer effects. XTT cell proliferation assay showed that colchicine inhibited the growth and proliferation of human hypopharyngeal cancer cells (FaDu and SNU1041) in a dose- and time-dependent manner. Colchicine also inhibited the migration, invasion, and adhesion of hypopharyngeal cancer cells in a dose-dependent manner. The levels of mRNA expression and activity of matrix metalloproteinase-9 (MMP9) and urokinase-type plasminogen activator (uPA) decreased after treatment with colchicine. Further investigation revealed that colchicine inhibited the phosphorylation of the FAK/SRC complex and paxillin. Tumor volume ratios in colchicine-treated mice (0.1 mg/kg, every 2 days for 14 days) increased less than in control mice. To our knowledge, this is the first report showing that colchicine can suppress cell invasion, migration, and adhesion through reduced expression of MMP9, the uPA system, and the FAK/SRC complex. Colchicine has the potential to prevent disease progression in hypopharyngeal cancer and may have application as an adjunctive treatment., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Circular RNAs (circRNAs), a novel class of endogenous noncoding RNAs, have been shown to have important roles in a number of diseases, including several types of cancers. We hypothesized that circRNAs are involved in the pathogenesis of hypopharyngeal squamous cell carcinoma (HSCC). To test our hypothesis, we initially compared the expression profiles of circRNAs in 4 paired HSCC and adjacent normal tissue samples by using a circRNA microarray. The microarray data showed that 2392 circRNAs, including 1304 upregulated and 1088 downregulated circRNA transcripts, were significantly dysregulated in the HSCC tissues. The 10 most dysregulated circRNAs from the microarray analysis were further validated in another 32 pairs of specimens using quantitative real-time polymerase chain reaction assays. These circRNAs might sponge microRNAs (miRNAs) in predicted circRNA-miRNA-mRNA networks. Bioinformatics analysis was also performed to predict possible pathways in which these networks might be involved. Finally, we analyzed the interaction between validated circRNAs and their potential cancer-related miRNA targets. We are the first to comprehensively delineate the expression profiles of circRNAs in HSCC and to provide potential candidates for future mechanism studies. Our study is potentially of critical significance in uncovering the roles of circRNAs in HSCC.

Background/aim: The role of microRNAs (miRNA) in carcinogenesis is related to their genome-regulatory function. The aim of the present study was to identify and compare miRNA expression signatures of meso- and hypopharynx squamous cell cancers in consideration of the cancer field hypothesis., Patients and Methods: Thirteen snap-frozen biopsy series of tumors and peritumoral tissues from the meso- and hypopharynx were analyzed regarding their miRNA expression with quantitative real-time polymerase chain reaction (qRT-PCR)., Results: Significant miRNA expression changes of miR-21, -27a, -34a, -143 and -146a were found in peritumoral tissues that were dependent from the tumor location and the distance from the primary tumor site., Conclusion: miRNA expression analysis was found to be appropriate for molecular segregation of tumor location and peritumoral tissue segments, and appears to be a promising marker for cancer field characterization., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Radioresistance remains a major problem in the treatment of patients with hypopharyngeal squamous cell carcinoma (HSCC). Long noncoding RNAs (lncRNAs) have important roles in the development, invasion, and metastasis of various tumors, including HSCC, but little is known about the role of lncRNAs in cancer radioresistance. The aim of this study was to identify radioresistance-related lncRNAs and mRNAs in radioresistant (RS) hypopharyngeal cancer subclone RS-FaDu cells. In this study, we performed microarray analysis to find the differences in time-course lncRNA and mRNA expression profiles between RS-FaDu and parent FaDu cells after 4 Gy radiation therapy, whose reliability was confirmed by validation experiment. Among these consistently dysregulated lncRNAs, we found that some lncRNAs (e.g., TCONS&#95;00018436) might control resistance of HSCC cells to radiation. Furthermore, our bioinformatics analyses from mRNA/lncRNA microarray data showed that certain lncRNAs or mRNAs potentially are involved in radioresistance of HSCC. Our results from this study laid the foundation for further investigating the roles of these lncRNAs and mRNAs as promising candidates in the occurrence and development of HSCC radioresistance.

Laryngeal/hypopharyngeal liposarcomas are very rare, fewer than 40 cases have been reported. These tumors are polypoid, with a male predisposition, and usually cause hoarseness and difficulty breathing. Their clinical course is characterized by multiple local recurrences. No distant metastasis has been reported, and dedifferentiation is extremely rare. In sum, the prognosis of these tumors is excellent; the 5-year survival rate is essentially 100 %. Pathologic diagnosis of these well-differentiated liposarcomas can be challenging. Many of them were initially diagnosed as benign lipoma, fibrovascular polyp, or "inflammatory polyp". The correct diagnosis is usually made after multiple recurrences. On the other hand, the literature bears out that these incorrect diagnoses do not impact disease-specific survival. Here, we report three patients with laryngeal/hypopharyngeal well-differentiated liposarcomas; this is the first documentation of MDM2 amplification in liposarcomas at this site.

Macrophages play a critical role in tumor invasion and metastasis, which remain major causes of mortality in patients with hypopharyngeal cancer. Here we investigate the effect of an oncogene, AEG-1 expressed in macrophages on the invasion of hypopharyngeal cancer cells. AEG-1 is more highly expressed in macrophages of human hypopharyngeal cancer samples compared with adjacent non-tumor controls. Using matrigel invasion assay system, THP-1-derived macrophages with forced AEG-1 overexpression enhance FaDu cell invasion whereas macrophages with AEG-1 silence inhibit. Matrix metalloproteinase 9 (MMP-9), which is important in tumor invasion and metastasis through degrading extracellular matrix, is up-reulated by AEG-1 partly through NF-κB p65 in macrophages. Intriguingly, macrophage AEG-1 also induces MMP-9 up-regulated expression in FaDu cells. Furthermore, macrophage AEG-1 activates signal transducer and activator of transcription 3 (STAT3) in FaDu cells, which is responsible for macrophage AEG-1-induced an increase in MMP-9 expression and invasion of FaDu cells. This is the first to demonstrate that macrophage AEG-1 promotes tumor invasion through up-regulation of MMP-9 in both macrophages and cancer cells. Thus, the results provide evidences that macrophage AEG-1 contributes to promotion of tumor invasion, and represents as a potential target in hypopharyngeal cancer therapy.

Background: The present study aims to further explore the role of STK33 in hypopharyngeal squamous cell carcinoma (HSCC), with special attention given to the possible relationship between STK33 alteration and calcium., Methods: An in vivo experiment and microarray analysis were performed to investigate the impact of STK33 knockdown (STK33-RNAi) on the biological behaviors and the gene profile alterations of a HSCC cell line (Fadu). Cell viability and morphological change of Fadu cells in response to Ionomycin were measured by MTT assay and acridine orange staining. The concentration of intracellular calcium ([Ca(2+)]i) was detected by laser scanning confocal microscope with fluo-3/AM. The mRNA and protein expressions of relevant genes were examined by real-time PCR and Western blot., Results: STK33-RNAi retarded the Fadu cell proliferation and the metastasis in nude mice and led to up- and down-regulation of the expressions of abundance of genes, especially, the downregulation of the CAPN1 gene. Ionomycin increased the [Ca(2+)]i and decreased the survival rates of Fadu cells in a time-dependent manner. Moreover, Ionomycin resulted in the elevation of CAPN1 mRNA expression in normal Fadu cells and, conversely, had almost no effect on CAPN1 expression in STK33-RNAi cells., Conclusions: Findings from this work further validate that STK33 is a potential oncogene and plays an important role in tumorigenesis of HSCC via regulation of numerous genes. In addition, there exists the reciprocal influence between STK33 and [Ca(2+)]i in Fadu cells.

Histone H3 (H3K4) demethylase JARID1B is aberrantly upregulated in many types of tumor and has been proposed to function as oncogene. Here we show that JARID1B is elevated in moderate and high-differentiated human hypopharyngeal squamous cell carcinoma (HPSCC) compared with low-differentiated HPSCC. Overexpression of JARID1B in FaDu cells increased epithelial differentiation marker K10 expression and inhibited cell proliferation. JARID1B and K10 mRNA expression is high correlated in HPSCC patients. Mechanistically, we found JARID1B directly bound to PI3K/AKT signaling inhibitor SHIP1 gene promoter and decreased SHIP1 gene expression. Activation of downstream AKT resulted in increased β-catenin signaling, by which promoted target genes Fra-1 and Jun, together with other AP-1 transcription factors, leading to K10 expression. Forced expression of SHIP1 rescued JARID1B-induced phenotypes on FaDu cell differentiation and proliferation. Taken together, our findings provide first evidence that elevated expression of JARID1B has a critical role in promoting HPSCC differentiation and inhibiting proliferation, suggesting JARID1B may function as a tumor suppressor in squamous cell cancers and implying a novel important therapeutic strategy of HPSCC.

Background: Selenium-binding protein 1 (SELENBP1) expression is reduced markedly in many types of cancers and low SELENBP1 expression levels are associated with poor patient prognosis., Methods: SELENBP1 gene expression in head and neck squamous cell carcinoma (HNSCC) was analyzed with GEO dataset and characteristics of SELENBP1 expression in paraffin embedded tissue were summarized. Expression of SELENBP1 in nasopharyngeal carcinoma (NPC), laryngeal cancer, oral cancer, tonsil cancer, hypopharyngeal cancer and normal tissues were detected using immunohistochemistry, at last, 99 NPC patients were followed up more than 5 years and were analyzed the prognostic significance of SELENBP1., Results: Analysis of GEO dataset concluded that SELENBP1 gene expression in HNSCC was lower than that in normal tissue (P < 0.01), but there was no significant difference of SELENBP1 gene expression in different T-stage and N-stage (P > 0.05). Analysis of pathological section concluded that SELENBP1 in the majority of HNSCC is low expression and in cancer nests is lower expression than surrounding normal tissue, even associated with the malignant degree of tumor. Further study indicated the low SELENBP1 expression group of patients with NPC accompanied by poor overall survival and has significantly different comparing with the high expression group., Conclusion: SELENBP1 expression was down-regulated in HNSCC, but has no associated with T-stage and N-stage of tumor. Low expression of SELENBP1 in patients with NPC has poor over survival, so SELENBP1 could be a novel biomarker for predicting prognosis., Competing Interests: The authors report no conflicts of interest.