DAZAP1 Phase Separation Regulates Mitochondrial Metabolism to Facilitate Invasion and Metastasis of Oral Squamous Cell Carcinoma.

Tumor invasion and metastasis are the underlying causes of the high mortality rate of oral squamous cell carcinoma (OSCC). Energy metabolism reprogramming has been identified as a crucial process mediating tumor metastasis, thus indicating an urgent need for in-depth investigation of the specific mechanisms of tumor energy metabolism. Here, we identified an RNA-binding protein, DAZ associated protein 1 (DAZAP1), as a tumor-promoting factor with an important role in OSCC progression. DAZAP1 was significantly upregulated in OSCC, which enhanced the migration and invasion of OSCC cells and induced the epithelial-mesenchymal transition (EMT). RNA-seq analysis and experimental validation demonstrated that DAZAP1 regulates mitochondrial energy metabolism in OSCC. Mechanistically, DAZAP1 underwent liquid-liquid phase separation (LLPS) to accumulate in the nucleus where it enhanced cytochrome-c oxidase 16 (COX16) expression by regulating pre-mRNA alternative splicing, thereby promoting OSCC invasion and mitochondrial respiration. In mouse OSCC models, loss of DAZAP1 suppressed EMT, downregulated COX16, and reduced tumor growth and metastasis. In OSCC patient samples, expression of DAZAP1 positively correlated with COX16, and high expression of both proteins was associated with poor patient prognosis. Together, these findings revealed a mechanism by which DAZAP1 supports mitochondrial metabolism and tumor development of OSCC, suggesting the potential of therapeutic strategies targeting DAZAP1 to block OSCC invasion and metastasis.

Neutrophil extracellular traps promote invasion and metastasis via NLRP3-mediated oral squamous cell carcinoma pyroptosis inhibition.

Neutrophil extracellular traps (NETs) are reticular structures composed of neutrophil elastase (NE), cathepsin G (CG) and DNA-histone enzyme complexes. Accumulating evidence has revealed that NETs play important roles in tumor progression, metastasis, and thrombosis. However, our understanding of its clinical value and mechanism of action in oral squamous cell carcinoma (OSCC) is limited and has not yet been systematically described. Here, we aimed to investigate the clinical significance of NETs in OSCC and the mechanisms by which they affect its invasive and metastatic capacity. Our results demonstrated that high enrichment of NETs is associated with poor prognosis in OSCC, and mechanistic studies have shown that NE in NETs promotes invasion and metastasis via NLRP3-mediated inhibition of pyroptosis in OSCC. These findings may provide a new therapeutic approach for OSCC.

Identification of immunogenic cell death-related damage-related molecular patterns (DAMPs) to predict outcomes in patients with head and neck squamous cell carcinoma.

PURPOSE: Head and neck cancer is the sixth most common type of cancer worldwide, wherein the immune responses are closely associated with disease occurrence, development, and prognosis. Investigation of the role of immunogenic cell death-related genes (ICDGs) in adaptive immune response activation may provide cues into the mechanism underlying the outcome of HNSCC immunotherapy. METHODS: ICDGs expression patterns in HNSCC were analyzed, after which consensus clustering in HNSCC cohort conducted. A 4-gene prognostic model was constructed through LASSO and Cox regression analyses to analyze the prognostic index using the TCGA dataset, followed by validation with two GEO datasets. The distribution of immune cells and the response to immunotherapy were compared between different risk subtypes through multiple algorithms. Moreover, immunohistochemical (IHC) analyses were conducted to validate the prognostic value of HSP90AA1 as a predictor of HNSCC patient prognosis. In vitro assays were performed to further detect the effect of HSP90AA1 in the development of HNSCC. RESULTS: A novel prognostic index based on four ICDGs was constructed and proved to be useful as an independent factor of HNSCC prognosis. The risk score derived from this model grouped patients into high- and low-risk subtypes, wherein the high-risk subtype had worse survival outcomes and poorer immunotherapy response. IHC analysis validated the applicability of HSP90AA1 as a predictor of prognosis of HNSCC patients. HSP90AA1 expression in tumor cells promotes the progression of HNSCC. CONCLUSIONS: Together, these results highlight a novel four-gene prognostic signature as a valuable tool to assess survival status and prognosis of HNSCC patients.

Tertiary lymphoid structures in head and neck squamous cell carcinoma improve prognosis by recruiting CD8+ T cells.

Tertiary lymphoid structures (TLSs) are formed in long-term chronic inflammation, promoting the local recruitment of lymphocytes, antigen presentation and regulation of immune response, correlated with a better prognosis for cancer patients. Although studies have been conducted to explore methods that accelerate the establishment of TLSs, related research in head and neck squamous cell carcinoma (HNSCC) is still lacking. In this study, we analysed data from The Cancer Genome Atlas and performed immunohistochemical staining analyses of 188 patient samples. The results showed that TLSs promoted the infiltration of immune cells. Patients with TLSs with high infiltration of CD8+ cells showed the best prognosis. Since lymphotoxin α (LTα) was significantly increased in tissues with TLSs, we overexpressed LTα in SCC7 cells (a mouse-derived HNSCC cell line) and established tongue-tumour-bearing models. The polychromatic observation of tissue sections showed that T-cell aggregation increased in the LTα cell group, and a grade 1 TLS was formed on the 12th day after inoculating the cells. Moreover, the tumour volume in the LTα group was significantly less than that of the control group, whereas both the number and the proportion of infiltrated CD8+ T cells were increased. The peripheral CD8+ cells in mice were removed, and no difference was observed in tumour size or TLS formation. Remarkably, we found that TLS led to an increase in the antitumour effect by recruiting CD8+ T cells in HNSCC, showing a CD8+ T-cell-dependent antitumour effect. Moreover, LTα overexpression in the tumour promoted the formation of TLSs.

Tertiary lymphoid structures in oral lichen planus and oral epithelial dysplasia with lichenoid features: A comparative study.

OBJECTIVE: Tertiary lymphoid structures (TLSs) provide sites for antigen presentation and activation of lymphocytes, promoting their infiltration; thus, enhancing specific immune responses. The aim of this comparative cross-sectional study was to reveal the characteristics and influence of TLSs in oral lichen planus (OLP) and oral epithelial dysplasia (OED) with lichenoid features. METHODS: Clinical information and samples of 51 OLP and 19 OED with lichenoid features were collected. Immunohistochemistry was performed, and the structures where CD20+ B cells and CD3+ T cells aggregated with peripheral lymph node addressin positive (PNAd+) vessels were defined as TLSs. The results and clinical information were analysed. RESULT: TLS were found in 44 (86.3%) patients with OLP and 19 (100%) patients with OED. The TLS score was higher in OED group (p = 0.023), accompanied by an increased number of PNAd+ vessels. The TLS was significantly correlated with PNAd+ vessels (p = 0.027), CD20+ B (p < 0.001) and CD208+ dendritic cells (p = 0.001). Foxp3+ Treg cells but not CD8+ T cells infiltrated more severely in OED (p = 0.003) and increased when TLS score was high (p = 0.002). CONCLUSIONS: This study revealed the widespread development of TLSs in the OLP and OED. The presence of TLSs showed a close relationship with dysplasia and may increase malignant potency by over-inducing Treg cells.

Capnocytophaga gingivalis is a potential tumor promotor in oral cancer.

OBJECTIVES: To investigate the role of oral microbiome in promoting oral squamous cell carcinoma (OSCC) development. MATERIALS AND METHODS: We investigated the salivary microbiome of 108 controls and 70 OSCC cases by16S rRNA gene sequencing and detected the fluorescence signal of OSCC-related pathological bacteria by fluorescence in situ hybridization assay (FISH). The invasion and migration assays were used to show the differences of invasive and migrative abilities between control and experimental groups. Quantitative real-time PCR and Western blotting were used to verify the epithelial-to-mesenchymal transition (EMT). RESULTS: In our study, the overall microbiome abundance and composition were richer in the 108 controls than in the 70 OSCC cases. We demonstrated that Streptococcus, Capnocytophaga, Peptostreptococcus, and Lactobacillus were highly abundant in the saliva of OSCC patients by 16S rDNA sequencing and FISH. Moreover, we found that Capnocytophaga gingivalis (C. gingivalis) was highly presented in OSCC tissues by FISH. We focused on C. gingivalis and found that its supernatant induced OSCC cells to undergo EMT, causing the cells to acquire a mesenchymal phenotype associated with highly invasive and metastatic properties. CONCLUSION: Taken together, these results indicated that C. gingivalis might invade OSCC tissues and played an important role in OSCC by promoting OSCC invasion and metastasis by inducing EMT. Hence, the role of C. gingivalis in cancer progression revealed a new direction for the research of OSCC.

lncRNA CYTOR promotes aberrant glycolysis and mitochondrial respiration via HNRNPC-mediated ZEB1 stabilization in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC), the most common malignancy of the oral and maxillofacial region, severely affects human health. However, current treatments for OSCC commonly show only a ~60% 5-year survival rate of patients with distant metastases, indicating an urgent need for targeted treatments for patients with advanced metastases. Here, we report a survival-related long non-coding RNA, CYTOR, which is highly expressed in the lesions of oral cancer patients. We found that CYTOR can promote both migration and invasion in oral cancer cells as well as the epithelial-mesenchymal transition (EMT). RNA-sequencing of CYTOR-knockdown oral cancer cells revealed that CYTOR can regulate mitochondrial respiration and RNA splicing. Mechanistically, we found that nuclear-localized CYTOR interacts with HNRNPC, resulting in stabilization of ZEB1 mRNAs by inhibiting the nondegradative ubiquitination of HNRNPC. By synthesizing CYTOR-targeting small interfering RNAs (siRNAs) encapsulated in Nanoscale Metal Organic Frameworks (NMOFs), we demonstrate the targeted suppression of CYTOR to inhibit invasion and metastasis of oral cancer cells in a nude mouse model. Cumulatively, this study reveals the potential role of the CYTOR-HNRNPC-ZEB1 axis in regulating mitochondrial metabolism and glycolysis of oral cancer cells, and illustrates the effective use of lncRNA targeting in anti-metastatic cancer therapies.

Development of a prognostic pyroptosis-related gene signature for head and neck squamous cell carcinoma patient.

OBJECTIVE: Head and neck squamous cell carcinoma (HNSCC) is a major threat to public health. Pyroptosis is a form of inflammatory programmed cell death that is still incompletely understood. The role of pyroptotic cell death in HNSCC remains to be fully defined. As such, the present study was developed to explore the potential prognostic utility of a pyroptosis-related gene (PRG) signature in HNSCC. METHODS: PRG expression patterns and the associated mutational landscape in HNSCC were analyzed, after which a 6-gene prognostic model was constructed through least absolute shrinkage and selection operator (LASSO) and Cox regression analyses using the TCGA dataset, followed by validation with two GEO datasets (GSE41643 and GSE65858). The relative expression of the genes in the prognostic model was assessed via RT-qPCR in tumor and paired adjacent normal tissue samples from a 32-patient cohort. Potential predictors of patient outcomes associated with this 6-gene model were identified through topological degree analyses of a protein-protein interaction network. Moreover, the prognostic value of NLRP3 as a predictor of HNSCC patient prognosis was established through immunohistochemical (IHC) analyses of samples from 176 HNSCC patients. Lastly, in vitro studies were performed to further demonstrate the relevance of NLRP3 in the context of HNSCC development. RESULTS: Differentially expressed PRGs were able to readily differentiate between HNSCC tumors and normal tissues. Risk scores derived from the 6-gene PRG model were independent predictors of HNSCC patient prognosis, and genes that were differentially expressed between low- and high-risk groups were associated with tumor immunity. RT-qPCR assays also showed the potential protective role of NLRP3 in HNSCC patients. IHC analyses further supported the value of NLRP3 as a predictor of HNSCC patient outcomes. Invasion and migration assays demonstrated the potential role of NLRP3 in the inhibition of HNSCC development. CONCLUSIONS: Overall, these results highlight a novel prognostic gene signature that offers value in the context of HNSCC patient evaluation, although additional research will be essential to elucidate the mechanisms linking these PRGs to HNSCC outcomes.

Cancer-associated fibroblasts promote oral squamous cell carcinoma progression through LOX-mediated matrix stiffness.

BACKGROUND: Cancer-associated fibroblasts (CAFs), the most abundant cells in the tumor microenvironment, have prominent roles in the development of solid tumors as stromal targets. However, the underlying mechanism of CAFs' function in oral squamous cell carcinoma (OSCC) development remains unclear. Here, we investigated the role of lysyl oxidase (LOX) expression in CAFs in tumor stromal remodeling and the mechanism of its effect on OSCC progression. METHODS: Multiple immunohistochemistry (IHC) staining was performed to detect the correlation of CAFs and LOX in the stroma of OSCC specimens, as well as the correlation with clinicopathological parameters and prognosis. The expression of LOX in CAFs were detected by RT-qPCR and western blot. The effects of LOX in CAFs on the biological characteristics of OSCC cell line were investigated using CCK-8, wound-healing and transwell assay. CAFs were co-cultured with type I collagen in vitro, and collagen contraction test, microstructure observation and rheometer were used to detect the effect of CAFs on remodeling collagen matrix. Then, collagen with different stiffness were established to investigate the effect of matrix stiffness on the progression of OSCC. Moreover, we used focal adhesion kinase (FAK) phosphorylation inhibitors to explored whether the increase in matrix stiffness promote the progression of OSCC through activating FAK phosphorylation pathway. RESULTS: LOX was colocalized with CAFs in the stroma of OSCC tissues, and its expression was significantly related to the degree of malignant differentiation and poor prognosis in OSCC. LOX was highly expressed in CAFs, and its knockdown impaired the proliferation, migration, invasion and EMT process of OSCC cells. The expression of LOX in CAFs can catalyze collagen crosslinking and increase matrix stiffness. Furthermore, CAFs-derived LOX-mediated increase in collagen stiffness induced morphological changes and promoted invasion and EMT process in OSCC cells by activating FAK phosphorylation pathway. CONCLUSIONS: Our findings suggest that CAFs highly express LOX in the stroma of OSCC and can remodel the matrix collagen microenvironment, and the increase in matrix stiffness mediated by CAFs-derived LOX promotes OSCC development through FAK phosphorylation pathway. Thus, LOX may be a potential target for the early diagnosis and therapeutic treatment of OSCC.