The Impact of m1A Methylation Modification Patterns on Tumor Immune Microenvironment and Prognosis in Oral Squamous Cell Carcinoma

N1-methyladenosine (m1A) modification widely participates in the occurrence and progression of numerous diseases. Nevertheless, the potential roles of m1A in the tumor immune microenvironment (TIME) are still not fully understood. Based on 10 m1A methylation regulators, we comprehensively explored the m1A modification patterns in 502 patients with oral squamous cell carcinoma (OSCC). The m1A modification patterns were correlated with TIME characteristics and the m1A score was established to evaluate the effect of the m1A modification patterns on individual OSCC patients. The TIME characteristics and survival outcomes under the three m1A modification patterns were significantly distinct. OSCC patients in the high m1A score group were characterized by poorer prognosis, lower immune infiltration, lower ssGSEA score, lower expression levels of immune checkpoint molecules, and higher tumor mutation loads. The present study revealed that m1A modification might be associated with the TIME in OSCC, and has potential predictive ability for the prognosis of OSCC.     

The Role of Deubiquitinating Enzyme in Head and Neck Squamous Cell Carcinoma

Ubiquitination and deubiquitination are two popular ways for the post-translational modification of proteins. These two modifications affect intracellular localization, stability, and function of target proteins. The process of deubiquitination is involved in histone modification, cell cycle regulation, cell differentiation, apoptosis, endocytosis, autophagy, and DNA repair after damage. Moreover, it is involved in the processes of carcinogenesis and cancer development. In this review, we discuss these issues in understanding deubiquitinating enzyme (DUB) function in head and neck squamous cell carcinoma (HNSCC), and their potential therapeutic strategies for HNSCC patients are also discussed.     

Effect of EGFR on SQSTM1 Expression in Malignancy and Tumor Progression of Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma (OSCC) is one of the most common types of malignant tumor. Sequestosome 1 (SQSTM1) serves as an adaptor of autophagy for degrading protein aggregates. The regulation of autophagy by EGFR and its clinical impacts are indicated in various types of cancer. However, the association of EGFR and SQSTM1 in OSCC is still unknown. Our results show that the expression levels of SQSTM1 and EGFR proteins are higher in tumor tissues than in the corresponding tumor-adjacent (CTAN) tissues of OSCC patients. The expression levels of SQSTM1 were positively associated with the EGFR expression level. High co-expression of SQSTM1 and EGFR is associated with poor prognosis in OSCC patients. Moreover, SQSTM1 expression is decreased in EGFR-knockdown cells. Cell growth and invasion/migration are also decreased in cells with single/combined knockdowns of EGFR and SQSTM1 or in SQSTM1-knockdown cells without EGFR kinase inhibitor Lapatinib treatment compared to that in scrambled cells. However, cell growth and invasion/metastasis were not significantly different between the scrambled cells and SQSTM1-knockdown cells in the presence of Lapatinib. This study is the first to indicate the biological roles and clinical significance of SQSTM1 regulation by EGFR in OSCC.     

EGFR Mutations in Head and Neck Squamous Cell Carcinoma

EGFR is a prototypical receptor tyrosine kinase that is overexpressed in multiple cancers including head and neck squamous cell carcinoma (HNSCC). The standard of care for HNSCC remains largely unchanged despite decades of research. While EGFR blockade is an attractive target in HNSCC patients and anti-EGFR strategies including monoclonal antibodies and kinase inhibitors have shown some clinical benefit, efficacy is often due to the eventual development of resistance. In this review, we discuss how the acquisition of mutations in various domains of the EGFR gene not only alter drug binding dynamics giving rise to resistance, but also how mutations can impact radiation response and overall survival in HNSCC patients. A better understanding of the EGFR mutational landscape and its dynamic effects on treatment resistance hold the potential to better stratify patients for targeted therapies in order to maximize therapeutic benefits.     

Prognostic and Predictive Factors in Advanced Head and Neck Squamous Cell Carcinoma

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease arising from the mucosa of the upper aerodigestive tract. Despite multimodality treatments approximately half of all patients with locally advanced disease relapse and the prognosis of patients with recurrent or metastatic HNSCC is dismal. The introduction of checkpoint inhibitors improved the treatment options for these patients and pembrolizumab alone or in combination with a platinum and fluorouracil is now the standard of care for first-line therapy. However, approximately only one third of unselected patients respond to this combination and the response rate to checkpoint inhibitors alone is even lower. This shows that there is an urgent need to improve prognostication and prediction of treatment benefits in patients with HNSCC. In this review, we summarize the most relevant risk factors in the field and discuss their roles and limitations. The human papilloma virus (HPV) status for patients with oropharyngeal cancer and the combined positive score are the only biomarkers consistently used in clinical routine. Other factors, such as the tumor mutational burden and the immune microenvironment have been highly studied and are promising but need validation in prospective trials.     

Clinical Impact of Tumor-Infiltrating Inflammatory Cells in Primary Small Cell Esophageal Carcinoma

Primary small cell esophageal carcinoma is a rare and aggressive type of gastrointestinal cancer with poor prognosis. In the present study, the impact of tumour infiltrating inflammatory cells on clinico-pathological characteristics and the patients’ prognosis were analysed. A total of 36 small cell esophageal carcinomas, 19 adjacent normal tissues and 16 esophageal squamous cell carcinoma samples were collected. Qualified pathologists examined eosinophils, neutrophils, lymphocytes and macrophages on histochemical slides. The infiltration of eosinophils and macrophages in small cell esophageal carcinoma was significantly increased as compared with tumor adjacent normal tissues, and was significantly less in esophageal squamous cell carcinoma. Macrophage count was significantly associated with (p = 0.015) lymph node—stage in small cell esophageal carcinoma. When we grouped patients into two groups by counts of infiltrated inflammatory cells, Kaplan-Meier analysis revealed that high macrophage infiltration group (p = 0.004) and high eosinophil infiltration group (p = 0.027) had significantly enhanced survival. In addition, multivariate analysis unveiled that eosinophil count (p = 0.002) and chemotherapy (Yes vs. No, p = 0.001) were independent prognostic indicators. Taken together, infiltration of macrophages and eosinophils into the solid tumor appear to be important in the progression of small cell esophageal carcinoma and patients’ prognosis.     

Salivary Cytokines as Biomarkers for Oral Squamous Cell Carcinoma: A Systematic Review

The prognosis of patients with oral squamous carcinoma (OSCC) largely depends on the stage at diagnosis, the 5-year survival rate being approximately 30% for advanced tumors. Early diagnosis, including the detection of lesions at risk for malignant transformation, is crucial for limiting the need for extensive surgery and for improving disease-free survival. Saliva has gained popularity as a readily available source of biomarkers (including cytokines) useful for diagnosing specific oral and systemic conditions. Particularly, the close interaction between oral dysplastic/neoplastic cells and saliva makes such fluid an ideal candidate for the development of non-invasive and highly accurate diagnostic tests. The present review has been designed to answer the question: “Is there evidence to support the role of specific salivary cytokines in the diagnosis of OSCC?” We retrieved 27 observational studies satisfying the inclusion and exclusion criteria. Among the most frequent cytokines investigated as candidates for OSCC biomarkers, IL-6, IL-8, TNF-α are present at higher concentration in the saliva of OSCC patients than in healthy controls and may therefore serve as basis for the development of rapid tests for early diagnosis of oral cancer.     

C2-Ceramide Induces Cell Death and Protective Autophagy in Head and Neck Squamous Cell Carcinoma Cells

Ceramides are second messengers involved in several intracellular processes in cancer cells, amongst others. The aim of this study was to evaluate the anti-tumor efficacy of C2-ceramide (C2-Cer; N-acetyl-d-sphingosine) by investigating cell death and autophagy in head and neck squamous cell carcinoma (HNSCC) cells. C2-Cer showed concentration-dependent cytotoxicity in HN4 and HN30 cell lines. It simultaneously induced caspase-3-independent apoptosis and programmed necrosis. C2-Cer markedly increased the expression level of microtubule-associated protein 1 light chain 3B (LC3B) type II associated with protective autophagy. An autophagy inhibitor enhanced C2-Cer-mediated cytotoxicity, while a programmed-necrosis inhibitor produced the opposite effect. Furthermore, C2-Cer up-regulated the phosphorylation of extracellular signal-regulated kinase 1/2, but down-regulated its downstream substrate phospho-mammalian target of rapamycin (p-mTOR) during the autophagy process. These results suggested that C2-Cer exerts anti-tumor effects by inducing programmed apoptosis and necrosis in HNSCC, and these cytotoxic effects are enhanced by an autophagy inhibitor.     

Role of Salivary MicroRNA and Cytokines in the Diagnosis and Prognosis of Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma (OSCC) is the most prevalent oral malignant tumor worldwide. An early diagnosis can have a major positive impact on its prognosis. Human saliva contains cytokines, DNA and RNA molecules, circulating cells, and derivatives of tissues and extracellular vesicles, among other factors that can serve as biomarkers. Hence, the analysis of saliva may provide useful information for the early diagnosis of OSCC for its prognosis. The objective of this review was to determine the potential usefulness of salivary biomarkers (cytokines and microRNA) to diagnose OSCC and improve its prognosis. A combination of salivary miRNA and proteomic data could allow a definitive and early diagnosis to be obtained. However, there remains a need to optimize and standardize the protocols used to quantify miRNAs.     

Epigenetic Deregulation of Protein Tyrosine Kinase 6 Promotes Carcinogenesis of Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma (OSCC) accounts for over 90% of oral cancers and causes considerable morbidity and mortality. Epigenetic deregulation is a common mechanism underlying carcinogenesis. DNA methylation deregulation is the epigenetic change observed during the transformation of normal cells to precancerous and eventually cancer cells. This study investigated the DNA methylation patterns of PTK6 during the development of OSCC. Bisulfite genomic DNA sequencing was performed to determine the PTK6 methylation level. OSCC animal models were established to examine changes in PTK6 expression in the different stages of OSCC development. The DNA methylation of PTK6 was decreased during the development of OSCC. The mRNA and protein expression of PTK6 was increased in OSCC cell lines compared with human normal oral keratinocytes. In mice, the methylation level of PTK6 decreased after treatment with 4-nitroquinoline 1-oxide and arecoline, and the mRNA and protein expression of PTK6 was increased. PTK6 hypomethylation can be a diagnostic marker of OSCC. Upregulation of PTK6 promoted the proliferation, migration, and invasion of OSCC cells. PTK6 promoted carcinogenesis and metastasis by increasing STAT3 phosphorylation and ZEB1 expression. The epigenetic deregulation of PTK6 can serve as a biomarker for the early detection of OSCC and as a treatment target.     

Proliferating Lymphatic Endothelial Cells as a Prognostic Marker in Head and Neck Squamous Cell Carcinoma

Podoplanin and Ki-67 are two important markers of cancer progression. The aim of this study is to evaluate double immunostaining for Ki-67 and podoplanin in head and neck squamous cell carcinoma (HNSCC), and to observe the involvement of lymphagiogenesis in tumoral and peritumoral areas, as well as the density of tumor proliferation correlated with histopathological grading. A total of 50 patients with HNSCC were included in this study. We carried out a morphological evaluation of tissue samples, after that, cases were selected for double Ki-67 and podoplanin immunostaining. Podoplanin expression was significantly correlated with histopathological grade (p < 0.05; p = 0.037) and expression of Ki-67 (p < 0.05; p = 0.050). A high expression of podoplanin, as well as of the proliferation factor Ki-67, was observed in histopathological grade G3 and the correlation between these (p < 0.05; p = 0.028), and implication of LMVD and LVI was not significant (LMVD p = 0.577; LVI p = 0.976). This study demonstrated the importance of double immunolabeling in assessing lymphagiogenesis and tumor proliferation in correlation with histopathological grades in HNSCC.     

In Situ PD-L1 Expression in Oral Squamous Cell Carcinoma Is Induced by Heterogeneous Mechanisms among Patients

The expression of programmed death ligand-1 (PD-L1) is controlled by complex mechanisms. The elucidation of the molecular mechanisms of PD-L1 expression is important for the exploration of new insights into PD-1 blockade therapy. Detailed mechanisms of the in situ expression of PD-L1 in tissues of oral squamous cell carcinomas (OSCCs) have not yet been clarified. We examined the mechanisms of PD-L1 expression focusing on the phosphorylation of downstream molecules of epidermal growth factor (EGF) and interferon gamma (IFN-γ) signaling in vitro and in vivo by immunoblotting and multi-fluorescence immunohistochemistry (MF-IHC), respectively. The in vitro experiments demonstrated that PD-L1 expression in OSCC cell lines is upregulated by EGF via the EGF receptor (EGFR)/PI3K/AKT pathway, the EGFR/STAT1 pathway, and the EGFR/MEK/ERK pathway, and by IFN-γ via the JAK2/STAT1 pathway. MF-IHC demonstrated that STAT1 and EGFR phosphorylation was frequently shown in PD-L1-positive cases and STAT1 phosphorylation was correlated with lymphocyte infiltration and EGFR phosphorylation. Moreover, the phosphorylation pattern of the related molecules in PD-L1-positive cells differed among the cases investigated. These findings indicate that PD-L1 expression mechanisms differ depending on the tissue environment and suggest that the examination of the tissue environment and molecular alterations of cancer cells affecting PD-L1 expression make it necessary for each patient to choose the appropriate combination drugs for PD-1 blockade cancer treatment.     

Progress of Nanomaterials-Based Photothermal Therapy for Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma (OSCC) is one of the top 15 most prevalent cancers worldwide. However, the current treatment models for OSCC (e.g., surgery, chemotherapy, radiotherapy, and combination therapy) present several limitations: damage to adjacent healthy tissue, possible recurrence, low efficiency, and severe side effects. In this context, nanomaterial-based photothermal therapy (PTT) has attracted extensive research attention. This paper reviews the latest progress in the application of biological nanomaterials for PTT in OSCC. We divide photothermal nanomaterials into four categories (noble metal nanomaterials, carbon-based nanomaterials, metal compounds, and organic nanomaterials) and introduce each category in detail. We also mention in detail the drug delivery systems for PTT of OSCC and briefly summarize the applications of hydrogels, liposomes, and micelles. Finally, we note the challenges faced by the clinical application of PTT nanomaterials and the possibility of further improvement, providing direction for the future research of PTT in OSCC treatment.     

Single-Cell RNA Sequencing Analysis for Oncogenic Mechanisms Underlying Oral Squamous Cell Carcinoma Carcinogenesis with Candida albicans Infection

Oral squamous cell carcinoma (OSCC) carcinogenesis involves heterogeneous tumor cells, and the tumor microenvironment (TME) is highly complex with many different cell types. Cancer cell–TME interactions are crucial in OSCC progression. Candida albicans (C. albicans)—frequently pre-sent in the oral potentially malignant disorder (OPMD) lesions and OSCC tissues—promotes malignant transformation. The aim of the study is to verify the mechanisms underlying OSCC car-cinogenesis with C. albicans infection and identify the biomarker for the early detection of OSCC and as the treatment target. The single-cell RNA sequencing analysis (scRNA-seq) was performed to explore the cell subtypes in normal oral mucosa, OPMD, and OSCC tissues. The cell composi-tion changes and oncogenic mechanisms underlying OSCC carcinogenesis with C. albicans infec-tion were investigated. Gene Set Variation Analysis (GSVA) was used to survey the mechanisms underlying OSCC carcinogenesis with and without C. albicans infection. The results revealed spe-cific cell clusters contributing to OSCC carcinogenesis with and without C. albicans infection. The major mechanisms involved in OSCC carcinogenesis without C. albicans infection are the IL2/STAT5, TNFα/NFκB, and TGFβ signaling pathways, whereas those involved in OSCC carcinogenesis with C. albicans infection are the KRAS signaling pathway and E2F target down-stream genes. Finally, stratifin (SFN) was validated to be a specific biomarker of OSCC with C. albicans infection. Thus, the detailed mechanism underlying OSCC carcinogenesis with C. albicans infection was determined and identified the treatment biomarker with potential precision medicine applications.     

Targeting mTOR-CCL20 Signaling May Improve Response to Docetaxel in Head and Neck Squamous Cell Carcinoma

Patients with advanced head and neck squamous cell carcinoma (HNSCC) usually show a dismal prognosis. It is this worthwhile to develop new, effective therapeutic regimens for these patients, such as molecular targeted therapy, which is promising as an alternative or combination treatment for HNSCC. The mammalian target of rapamycin (mTOR) pathway, which plays an important role in the carcinogenesis of HNSCC, is the most frequently activated, and is thus worthy of further investigation. In this study, two human HNSCC cell lines, FaDu and SAS, were evaluated for cell growth with trypan blue staining and tumor growth using an orthotopic xenograft model. The immunohistochemical expression of mTOR in the subcutaneous xenograft model and the inhibitory effects of docetaxel on the growth and state of activation of the PI3K/mTOR pathway were also evaluated and examined by colony formation and Western blot, respectively. Cell proliferation and migration were measured by water-soluble tetrazolium salt (WST-1) and Oris^TM cell migration assay, respectively. Furthermore, the effects of rapamycin and BEZ235, a phosphatidylinositol 3-kinases (PI3K) and mTOR inhibitor in combination with docetaxel or CCL20 were evaluated in the FaDu and SAS cells. The results showed that the expression of mTOR was significantly higher in the SAS and FaDu xenograft models than in the control. Docetaxel treatment significantly suppressed HNSCC cell proliferation and migration in vitro via the PI3K/mTOR/CCL-20 signaling pathway. Additionally, when administered in a dose-dependent fashion, mTOR inhibitors inhibited the growth and migration of the HNSCC cells. This combination was synergistic with docetaxel, resulting in almost complete cell growth and migration arrest. In conclusion, docetaxel significantly inhibited HNSCC cell proliferation and migration in vitro via the PI3K/mTOR/CCL-20 signaling pathway. The synergistic and additive activity of mTOR inhibitors combined with docetaxel shows potential as a new treatment strategy for HNSCC.     

Cinobufagin Exerts Anticancer Activity in Oral Squamous Cell Carcinoma Cells through Downregulation of ANO1

Anoctamin1 (ANO1), a calcium-activated chloride channel, is frequently overexpressed in several cancers, including oral squamous cell carcinoma (OSCC). OSCC is a highly aggressive cancer and the most common oral malignancy. ANO1 has been proposed as a potential candidate for targeted anticancer therapy. In this study, we performed a cell-based screening to identify novel regulators leading to the downregulation of ANO1, and discovered cinobufagin, which downregulated ANO1 expression in oral squamous cell carcinoma CAL-27 cells. ANO1 protein levels were significantly reduced by cinobufagin in a dose-dependent manner with an IC₅₀ value of ~26 nM. Unlike previous ANO1 inhibitors, short-term (≤10 min) exposure to cinobufagin did not alter ANO1 chloride channel activity and ANO1-dependent intestinal smooth muscle contraction, whereas long-term (24 h) exposure to cinobufagin significantly reduced phosphorylation of STAT3 and mRNA expression of ANO1 in CAL-27 cells. Notably, cinobufagin inhibited cell proliferation of CAL-27 cells expressing high levels of ANO1 more potently than that of ANO1 knockout CAL-27 cells. In addition, cinobufagin significantly reduced cell migration and induced caspase-3 activation and PARP cleavage in CAL-27 cells. These results suggest that downregulation of ANO1 by cinobufagin is a potential mechanism for the anticancer effect of cinobufagin in OSCC.     

The Multiple Roles of CD147 in the Development and Progression of Oral Squamous Cell Carcinoma: An Overview

Cluster of differentiation (CD)147, also termed extracellular matrix metalloprotease inducer or basigin, is a glycoprotein ubiquitously expressed throughout the human body, the oral cavity included. CD147 actively participates in physiological tissue development or growth and has important roles in reactive processes such as inflammation, immunity, and tissue repair. It is worth noting that deregulated expression and/or activity of CD147 is observed in chronic inflammatory or degenerative diseases, as well as in neoplasms. Among the latter, oral squamous cell carcinoma (OSCC) is characterized by an upregulation of CD147 in both the neoplastic and normal cells constituting the tumor mass. Most interestingly, the expression and/or activity of CD147 gradually increase as healthy oral mucosa becomes inflamed; hyperplastic/dysplastic lesions are then set on, and, eventually, OSCC develops. Based on these findings, here we summarize published studies which evaluate whether CD147 could be employed as a marker to monitor OSCC development and progression. Moreover, we describe CD147-promoted cellular and molecular events which are relevant to oral carcinogenesis, with the aim to provide useful information for assessing whether CD147 may be the target of novel therapeutic approaches directed against OSCC.