Methylation related genes are associated with prognosis of patients with head and neck squamous cell carcinoma via altering tumor immune microenvironment.

Background/purpose: Analysis of methylomes may enable prognostic stratification in patients with head and neck squamous cell carcinoma (HNSCC). This study aimed to identify methylation-related differentially expressed genes (mrDEGs), and to assess their efficacy in predicting patients' survival, tumor immune microenvironment alterations and immune checkpoints in patients with HNSCC. Materials and methods: The methylome and transcriptome data of 528 HNSCC and 50 normal samples from TCGA database were used as training cohort. We identified mrDEGs and constituted a risk score model using Kaplan-Meier analysis and multivariate Cox regression. The prognostic efficacy of the risk score was validated in GSE65858 and GSE41613. We determined the enrichment of previously defined biological processes of mrDEGs. We separated the HNSCC patients into low-risk and high-risk groups and compared their immune cell infiltration and immune checkpoints' expressions. Results: The risk score model was constituted by nine prognostic mrDEGs, including LIMD2, SYCP2, EPHX3, UCLH1, STC2, PRAME, SLC7A4, PLOD2, and ACADL. The risk score was a significant prognostic factor both in training (P < 0.001) and validation dataset (GSE65858: P = 0.008; GSE41613 = 0.015). The prognostic mrDEGs were enriched in multiple immune-associated pathways. Effector immune cells were increased in low-risk patients, including CD8+ T cells, activated CD4+ T cells, and plasma cells, whereas tumor associated M2 macrophages were recruited in the high-risk group. Expressions of immune checkpoints were generally higher in low-risk patients, including CTLA-4, PD-1 and LAG3. Conclusion: The mrDEGs can stratify HNSCC patients' prognosis, which correlates with alterations in tumor immune infiltrations and immune checkpoints.

Prognostic and therapeutic prediction by screening signature combinations from transcriptome-methylome interactions in oral squamous cell carcinoma.

DNA methylation pattern in oral squamous cell carcinoma (OSCC) remains poorly described. This study aimed to perform a genome-wide integrated analysis of the transcriptome and methylome and assess the efficacy of their prognostic signature model in patients with OSCC. We analyzed transcriptome and methylome data from 391 OSCC samples and 41 adjacent normal samples. A total of 8074 differentially expressed genes (DEGs) and 10,084 differentially expressed CpGs (DMCpGs) were identified. Then 241 DEGs with DMCpGs were identified. According to the prognostic analysis, the prognostic signature of methylation-related differentially expressed genes (mrDEGPS) was established. mrDEGPS consisted of seven prognostic methylation-related genes, including ESRRG, CCNA1, SLC20A1, COL6A6, FCGBP, CDKN2A, and ZNF43. mrDEGPS was a significant stratification factor of survival (P < 0.00001) irrespective of the clinical stage. The immune effector components, including B cells, CD4+ T cells, and CD8+ T cells, were decreased in the tumor environment of patients with high mrDEGPS. Immune checkpoint expressions, including CTLA-4, PD-1, LAG3, LGALS9, HAVCR2, and TIGHT, were comprehensively elevated (P < 0.001). The estimated half-maximal inhibitory concentration difference between low- and high-risk patients was inconsistent among chemotherapeutic drugs. In conclusion, the transcriptome-methylome interaction pattern in OSCC is complex. mrDEGPS can predict patient survival and responses to immunotherapy and chemotherapy and facilitate clinical decision-making in patients with OSCC.

Transcriptional and H3K27ac related genome profiles in oral squamous cell carcinoma cells treated with metformin.

Objectives: Metformin, a first-line drug that has been used for type 2 diabetes treatment, recently attracts broad attention for its therapeutic effects on diverse human cancers. However, its effect and the underlying mechanisms on oral squamous cell carcinoma (OSCC) are not well known. Materials and Methods: OSCC cells were used to evaluate the effect of metformin on cell proliferation and colony formation in vitro. Tumor formation assay in nude mice was conducted to assess the effect of metformin in vivo. Western blotting and immunohistochemistry stain were performed to investigate the effect of metformin on the expression of acetylation at lysine 27 of histone H3 (H3K27ac) and methylation at lysine 27 of histone H3 (H3K27me3) in vitro and in vivo. RNA-seq and ChIP-seq were performed to explore the genome profile to metformin treatment in OSCC cells. Results: Metformin inhibited OSCC cell proliferation and colony formation in vitro, as well as OSCC growth in vivo. Metformin increased the global H3K27ac modification in vitro. Transcriptome analysis suggested that metformin mainly downregulated pluripotency stem cell pathway, development involved pathways and upregulated cytokine and inflammatory pathways. Additionally, H3K27ac was involved in transcription, DNA repair and replication in metformin-treated OSCC cells. Conclusions: Metformin inhibits OSCC growth concomitant upregulated global level of H3K27ac in vitro. This study provides insights into the molecule and epigenome basis on application of metformin in OSCC treatment, and highlights the underlying mechanisms of reprogrammed cancer regulation and epigenetic histone modification.

A TP53 mutation model for the prediction of prognosis and therapeutic responses in head and neck squamous cell carcinoma.

BACKGROUND: Tumor protein p53 (TP53) is the most frequently mutated gene in head and neck squamous cell carcinoma (HNSC), and TP53 mutations are associated with inhibited immune signatures and poor prognosis. We established a TP53 mutation associated risk score model to evaluate the prognosis and therapeutic responses of patients with HNSC. METHODS: Differentially expressed genes between patients with and without TP53 mutations were determined by using data from the HNSC cohort in The Cancer Genome Atlas database. Patients with HNSC were divided into high- and low-risk groups based on a prognostic risk score that was generated from ten TP53 mutation associated genes via the multivariate Cox regression model. RESULTS: TP53 was the most common mutant gene in HNSC, and TP53 mutations were associated with immunogenic signatures, including the infiltration of immune cells and expression of immune-associated genes. Patients in the high-risk group had significantly poorer overall survival than those in the low-risk group. The high-risk group showed less response to anti-programmed cell death protein 1 (PD-1) therapy but high sensitivity to some chemotherapies. CONCLUSION: The risk score based on our TP53 mutation model was associated with poorer survival and could act as a specific predictor for assessing prognosis and therapeutic response in patients with HNSC.

Multiple congenital granular cell tumours of the maxilla and mandible: a rare case report and review of the literature.

Congenital granular cell tumour (CGCT) is a benign lesion that predominantly arises from the alveolar ridges of neonates, especially the maxilla. However, it's only 10 percent of multiple lesions in all reported cases, in which simultaneously mandibular and maxillary involvements are more extremely rare. For treatments of multiple CGCTs, few standard procedures were reported. In addition to surgical excision, which refers to a preferred method, conservative treatment is an available choice. Here, a case of multiple CGCTs using different therapeutic strategies was reported because of its rarity and innovation. A five-day-old female newborn presented two congenital masses attached to the right mandibular and maxillary alveolar ridge. The size of the mandibular lesion causing difficulty in feeding was 3 cm in diameter and 0.5 cm in the maxilla. Based on different manifestations, surgical excision and conservative treatment were adopted respectively. The mandibular mass was excised while that in the maxilla underwent spontaneous regression. Satisfactory results were achieved for this patient. There was no evidence of recurrence after a 6-month follow-up. Microscopic examination and immunohistochemistry analysis confirmed the diagnosis and differential diagnosis of CGCT and even proposed the possibility of histogenesis from neural crest. Moreover, we reviewed the literature and summarized the characteristics to provide new ideas for the treatment of multiple CGCTs.

Low expression of RalGAPs associates with the poorer overall survival of head and neck squamous cell carcinoma.

BACKGROUND: The role of Ral and RalGAPs on the progression of head and neck squamous cell carcinoma (HNSC) remains unclear. METHODS: The predesigned siRNAs against RalGAPs were transfected into cells to evaluate the effect on RalA activation. The Data from TCGA and GTEx were combined to analyze the pan-cancer gene expression of RalA and RalGAPs in cancer and adjacent normal tissues. Kaplan-Meier analysis was used to assess the predictive value of RalA and RalGAPs expression on the overall survival of patients with HNSC. Methylation-specific PCR in vitro and next-generation bisulfite sequencing in vivo were used to evaluate the association between DNA methylation and the down-regulation of RalGAPs. RESULTS: RalGAPs negatively regulated RalA activation. HNSC patients with low level of RalGAPα2 had worse overall survival. The promoter of RalGAPα2 was widely methylated in comparison to RalGAPα1 and the DNA methylation level of RalGAPα2 promoter was increased in HNSC tissues and associated with the presence of neck lymph node metastasis. CONCLUSIONS: RalA and RalGAPs could act as a specific predictor to assess the prognosis of HNSC. DNA methylation might be a potential mechanism that downregulated the RalGAPα2 expression.

[Bioinformatics analysis of programmed cell death ligand 1 co-expression genes and their regulatory network in head and neck squamous cell carcinoma].

OBJECTIVE: This study aimed to construct a network of programmed celldeath ligand 1 (PD-L1) co-expression genes and screen potential biomarkers for PD-L1 expression in head and neck squamous cell carcinoma (HNSCC) by bioinformatics analysis. Moreover, the genes and pathways participating in PD-L1 and regulating the tumor immune status were determined. METHODS: The HNSCC transcriptomic dataset in TCGA was selected to retrieve gene sets on the cBioPortal platform, where PD-L1 co-expressional genes were acquired. With these genes, GO-BP, KEGG, and string analyses were performed in R clusterProfiler. Cytoscape was used for network analysis and hub gene screening. RESULTS: A total of 117 co-expression genes were obtained, most of which were enriched in immune regulation and response to viral processes. Node degree analysis indicated that STAT1, IFNG, CXCL10, CCR5, FCGR3A, CXCL9, GBP5, CD86, GZMB, IRF1 were the highest connected genes and functioned as hub genes. Survival analysis of these hub genes resulted in CCR5, CXCL9, and GZMB as the prognostic biomarkers for patients with HNSCC, all of which were involved in immune regulation and their expression levels were related to PD-L1 (Pearson correlation coefficient was 0.30, 0.35, 0.39; P<0.01). High expression levels of these three hub genes were protective factors in patients with HNSCC. CONCLUSIONS: PD-L1 co-expression hub genes are related to immunity, among which CCR5, CXCL9, and GZMB are prognostic markers with the possibility to be involved in programmed cell death protein 1 (PD-1)/PD-L1-induced tumor immune escape. These genes provide new clues to study the mechanism and precision target medicine of PD-1/PD-L1 in HNSCC.

Exogenous VEGF introduced by bioceramic composite materials promotes the restoration of bone defect in rabbits.

This study aimed to investigate the effect of exogenous vascular endothelial growth factor (VEGF) introduced by bioceramic composite materials on jawbone defect. Rabbits were randomly divided into four groups: control, sham, model, and stent. In the model group, holes of jawbone defect were created through surgery. In the stent group, rabbits with jawbone defect were treated with polyether ketone (PEK)/biphasic bioceramic ((PEK-BBC)) composite materials encapsulating VEGF. At 4, 8, and 16 weeks post-operation, HE and Van Gieson staining of jawbones were performed to characterize the repair status of the bone defect. For all time intervals, we found intact bone structures in the control and sham groups and there was no improvement in the bone defect position in the model group. However, in the stent group, we excitingly observed the growth of many osteocytes in the margin of stents at 8 and 16 weeks. RT-PCR, western blot, and immunofluorescence analysis were conducted to investigate the VEGF expression at 4, 8, and 16 weeks post-operation. At 8 weeks, the level of VEGF in the model group was sharply downregulated as compared with the control group (P < .05) and interestingly, the stent group had a much higher level of VEGF than the model group (P < .05). At 16 weeks, the VEGF expression in the model group was further reduced comparing to the control group (P < .05), which was also elevated to a relative high level by the stent treatment (P < .05). As for the sham group, the VEGF level was stable without any difference from the control group at all time intervals. Therefore, exogenous VEGF introduced by bioceramic composite materials promoted the restoration of bone defect in rabbits.