Normal Oral Tissues Research Articles

Overexpression of a disintegrin and metalloproteinase 9 (ADAM9) in relation to poor prognosis of patients with oral squamous cell carcinoma

This study investigates the expressions of ADAM9, CDCP1 and t-PA in OSCC and their impacts on patient prognosis. Previous research has demonstrated the overexpression of ADAM9 and activation of plasminogen activator in OSCC, but CDCP1's role remains unexplored. While these biomolecules are known to contribute to lung cancer metastasis, their concurrent expressions in OSCC have not been thoroughly examined. Our aim is to assess the expressions of ADAM9, CDCP1, and t-PA in OSCC specimens, compare them with normal oral tissues, and explore their correlation with OSCC's clinicopathological features and patient survival outcomes.

Development and Characterization of a Three-Dimensional Organotypic In Vitro Oral Cancer Model with Four Co-Cultured Cell Types, Including Patient-Derived Cancer-Associated Fibroblasts.

Background/Objectives: Cancer organoids have emerged as a valuable tool of three-dimensional (3D) cell cultures to investigate tumor heterogeneity and predict tumor behavior and treatment response. We developed a 3D organotypic culture model of oral squamous cell carcinoma (OSCC) to recapitulate the tumor-stromal interface by co-culturing four cell types, including patient-derived cancer-associated fibroblasts (PD-CAFs). Methods: A stainless-steel ring was used twice to create the horizontal positioning of the cancer stroma (adjoining normal oral mucosa connective tissue) and the OSCC layer (surrounding normal oral mucosa epithelial layer). Combined with a structured bi-layered model of the epithelial component and the underlying stroma, this protocol enabled us to construct four distinct portions mimicking the oral cancer tissue arising in the oral mucosa. Results: In this model, α-smooth muscle actin-positive PD-CAFs were localized in close proximity to the OSCC layer, suggesting a crosstalk between them. Furthermore, a linear laminin-γ2 expression was lacking at the interface between the OSCC layer and the underlying stromal layer, indicating the loss of the basement membrane-like structure. Conclusions: Since the specific 3D architecture and polarity mimicking oral cancer in vivo provides a more accurate milieu of the tumor microenvironment (TME), it could be crucial in elucidating oral cancer TME.

PPT1 Promotes Growth and Inhibits Ferroptosis of Oral Squamous Cell Carcinoma Cells.

Oral squamous cell carcinoma (OSCC) is one of the most prevalent cancers with poor prognosis in the head and neck. Elucidating molecular mechanisms underlying OSCC occurrence and development is important for the therapy. Dysregulated palmitoylation-related enzymes have been reported in several cancers but OSCC. To explore the role of palmitoyl-protein thioesterase 1 (PPT1) in OSCC. Differentially expressed genes (DEGs) and related protein-protein interaction networks between normal oral epithelial and OSCC tissues were screened and constructed via different online databases. Tumor samples from 70 OSCC patients were evaluated for the relationship between PPT1 expression level and patients'clinic characteristics. The role of PPT1 in OSCC proliferation and metastasis was studied by functional experiments including MTT, colony formation, EdU incorporation and transwell assays. Lentivirus-based constructs were used to manipulate gene expression. FerroOrange probe and malondialdehyde assay were used to determine ferroptosis. Growth of OSCC cells in vivo was investigated by a xenograft mouse model. A total of 555 DEGs were obtained, and topological analysis revealed that PPT1 and GPX4 might play critical roles in OSCC. Increased PPT1 expression was found to be correlated with poor prognosis of OSCC patients. PPT1 effectively promoted the proliferation, migration and invasion while inhibited the ferroptosis of OSCC cells. PPT1 affected the expression of glutathione peroxidase 4 (GPX4). PPT1 promoted growth and inhibited ferroptosis of OSCC cells. PPT1 might be a potential target for OSCC therapy.

Expression and biological significance of topoisomerase II α (TOP2A) in oral squamous cell carcinoma

BackgroundTopoisomerase II α(TOP2A) is usually highly expressed in rapidly proliferating cells, and its expression is regulated by cell cycle. The relationship between TOP2A and oral squamous cell carcinoma (OSCC) needs further study.MethodsTOP2A immunoreactivity was analyzed using immunohistochemical (IHC) staining analysis in specimens from 20 OSCC patients. Based on the high-throughput sequencing and gene microarray database, the expression of TOP2A mRNA in OSCC was calculated and its ability to identify OSCC tissues was evaluated by diagnostic analysis. CRISPR screen was used to select the genes necessary for OSCC cell growth, and the gene set was analyzed for function enrichment. Single-cell RNA sequencing analysis was conducted to evaluate the expression level of TOP2A mRNA in OSCC cells.ResultsCompared with normal oral tissues, the expression of TOP2A protein was up-regulated in OSCC tissues. A total of 1240 OSCC and 428 non-OSCC oral tissue samples were included based on high-throughput dataset retrieval, and it was confirmed that TOP2A mRNA was highly expressed in OSCC tissues [SMD = 1.51 (95% CI 0.94–2.07), sROC AUC = 0.96 (95% CI 0.94–0.98)]. As an essential gene for OSCC cell growth, single-cell RNA sequencing data also confirmed that TOP2A mRNA expression was up-regulated in OSCC cells.ConclusionThe up-regulation of TOP2A may play a pivotal role in OSCC.

Comprehensive analysis of an mRNA co-expression network and a ceRNA network reveals potential prognostic biomarkers in oral squamous cell carcinoma

BackgroundOral squamous cell carcinoma (OSCC) is a prevalent and aggressive oral cancer with a poor prognosis. Its polygenic risk is likely influenced by complex transcriptional disorders involving networks of co-expressed and functionally related genes, though such investigations are limited in OSCC.MethodsWe analyzed the GSE37991 dataset, comprising 40 OSCC and 40 normal oral tissue samples from the Gene Expression Omnibus. Tumor-specific modules were identified using weighted correlation network analysis (WGCNA), leading to the selection of hub mRNAs and lncRNAs. These lncRNAs were used to construct lncRNA–mRNA and competing endogenous RNA networks. We further examined the expression profiles and survival data of these genes from the Cancer Genome Atlas. Prognostic markers were identified and validated through 5-year survival analysis and Cox proportional hazards modeling. RT-qPCR was used to validate the expression levels in clinical OSCC tissues.ResultsWe identified 1847 differentially expressed genes in OSCC tissues. WGCNA revealed four OSCC-specific modules, screening 120 hub mRNAs and five hub lncRNAs. Two prognostic markers (AQP5, IL-26) from hub mRNAs and three (FRMD5, INHBB, GUCY1A3) from the lncRNA–mRNA network were associated with survival. Validation showed lower expression of AQP5 and GUCY1A3, and higher expression of FRMD5 and INHBB in OSCC compared to normal tissues.ConclusionThis study enhances our understanding of transcriptional dysregulation in OSCC and may highlights AQP5, IL-26, FRMD5, INHBB, and GUCY1A3 as promising prognostic biomarkers.

Serum CXCL13 as a Novel Biomarker in Oral Squamous Cell Carcinoma.

Despite its low sensitivity (approximately 30%), squamous cell carcinoma (SCC) antigen is commonly utilized as a serum tumor marker for oral SCC (OSCC) in clinical settings. The objective of this research was to identify novel biomarkers for OSCC. Initially, we performed microarray analysis to evaluate the gene expression signatures of primary OSCC and normal oral mucosal tissues. Our findings showed the C-X-C motif chemokine ligand 13 (CXCL13) to be a promising novel biomarker as it was consistently overexpressed in primary OSCC tissues, a conclusion corroborated by polymerase chain reaction results. Subsequently, we measured serum CXCL13 levels in 125 patients with OSCC using a sandwich enzyme-linked immunosorbent assay and compared the results with those of 29 healthy individuals. Remarkably, the levels of serum CXCL13 were consistently elevated in patients with OSCC, and the high expression of serum CXCL13 was notably associated with tumor size and neck lymph node metastasis. Patients with advanced OSCC with high-serum CXCL13 levels exhibited poor prognosis regarding both overall and disease-free survival. Finally, spatial transcriptome analysis revealed CXCL13 and CD8 expressions within tumor area clusters but not in adjacent normal areas, suggesting specific overexpression of CXCL13 in primary OSCC tissues. These findings imply that serum CXCL13 holds diagnostic and prognostic value, showing promise as a novel biomarker for OSCC.

Histomorphometric Analysis of Oral Squamous Cell Carcinoma, Apparently Normal Adjacent Mucosa and Epithelial Dysplasia.

Oral squamous cell carcinoma (OSCC) comprises more than 90% of oral cavity cancer and remains the leading cause of death in oral disease. Limited studies have been conducted to evaluate cellular histomorphometry changes in OSCC compared to premalignant lesions such as Dysplastic leukoplakia (DL), Nondysplastic leukoplakia (NDL), and normal epithelial. This cross-sectional descriptive-analytical study was conducted on total 72 samples, including superficial areas of squamous cell carcinoma (SCCSF), Invasive Front of Squamous Cell Carcinoma (SCCIF), Apparently Normal Adjacent Oral Mucosa (SCCANM) or normal margin, Dysplastic leukoplakia (DL), Nondysplastic leukoplakia (NDL), and normal oral mucosa tissue (NOM) (N = 12 per group). ANOVA was used to compare the nucleus-to-cytoplasm ratio (N/C), nucleus area (NA), and cellular area (CA) of the stained hematoxylin and eosin (H&E) samples in the studied groups. A P value less than 0.05 was considered to be a significant level. There was a significant increase in the CA, NA, and N/C in the basal and parabasal layers from normal epithelium to dysplastic epithelium and OSCC. The highest NA, CA, and N/C were in the SCCIF and SCCSF groups, respectively, and the lowest was observed in NOM. In addition, SCCANM basal and parabasal layer cells had a significant difference in N/C compared to NOM, which indicates a high risk of SCCANM transformation into malignancy. Cell histomorphometry changes were observed from normal tissue to premalignant lesions and OSCC. These parameters can be used as indicators of the potential for transformation into malignancy in premalignant lesions.

BCAT1 promotes cell proliferation, migration, and invasion via the PI3K-Akt signaling pathway in oral squamous cell carcinoma.

To analyze the expression, biological function of branched chain amino-acid transaminase 1 (BCAT1) in oral squamous cell carcinoma (OSCC). Real-time PCR and immunohistochemistry were used to analyze the expression of BCAT1 protein in OSCC and normal oral tissues. Based on the clinicopathological information of patients, the relationship between the expression of BCAT1 protein and other clinicopathological factors was analyzed. Real-time PCR and western blot assays were used to analyze the expression of BCAT1 gene and protein in normal human oral keratinocytes (HOK) and human OSCC cells, respectively. After BCAT1 overexpression or knockdown, the proliferation, cell cycle, migration, and invasion of human OSCC cells were analyzed by CCK8, flow cytometry, wound healing, and transwell invasion assays, respectively. After adding the BCAT1 inhibitor EGR240 to OSCC cells, the changes in cell proliferation, migration, and invasion ability in OSCC cells were analyzed. Based on the TCGA database, the involved signal pathway in BCAT1-related and BCAT1-binding genes was obtained for Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, verified by western blot assays. After inhibiting PI3K, the effect of BCAT1 on the expression of the downstream phosphorylated protein of the PI3K-Akt signaling pathway was analyzed by western blot assays. The relationship between the expression of BCAT1 and EMT-related protein of OSCC cells was also analyzed. The expression of BCAT1 gene and protein were upregulated in OSCC tissue, which positively correlated with the pathological grade of patients with OSCC. Compared with normal oral keratinocytes, BCAT1 gene and protein were upregulated in OSCC cells. BCAT1 overexpression promoted the proliferation, migration, and invasion of OSCC cells. BCAT1 knockdown or inhibition could reduce the proliferation, migration, and invasion abilities of OSCC cells. The results of bioinformatics analysis and Western bolt showed that BCAT1 could regulate the activation of PI3K-Akt signaling pathway, and promote epithelial-mesenchymal transition (EMT) of OSCC cells. BCAT1 could promote the proliferation, migration, and invasion of OSCC cells via PI3K-Akt signaling pathway, which is a potential therapeutic target for OSCC.

Galectin-3-upregulated FAK promotes angiogenesis through oral lichen planus-activated fibroblasts.

The specific mechanism underlying the role of oral lichen planus-activated fibroblasts in angiogenesis remains undefined. Herein, the expression of Galectin-3 in oral lichen planus and verifying whether Galectin-3 can promote angiogenesis through oral lichen planus-activated fibroblasts has been investigated. The expression of Galectin-3 and CD34 in the oral lichen planus tissues (n = 30) and normal oral mucosa tissues (n = 15) was detected by immunohistochemistry. The expression of Galectin-3 in the oral lichen planus-activated fibroblasts was determined by reverse transcription-polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay. Galectin-3 overexpression lentiviral vector was constructed and transfected with oral lichen planus-activated fibroblasts. In addition, oral lichen planus-activated fibroblasts were treated with GB1107 (5 and 10 μM) to inhibit Galectin-3 expression and co-cultured with human umbilical vein vascular endothelial cells, and analyzed by Transwell and tube formation assays. The expression of VEGF and FGF2 in oral lichen planus-activated fibroblasts was detected, and the expression and phosphorylation levels of VEGFR2 and FAP in human umbilical vein vascular endothelial cells were determined. Oral lichen planus subcutaneous tissues highly expressed Galectin-3, positively correlated with angiogenesis. Oral lichen planus-activated fibroblasts expressed significantly higher Galectin-3 than NFs. Oral lichen planus-activated fibroblasts overexpressing Galectin-3 enhanced the migration and tube-forming capacity of co-cultured human umbilical vein vascular endothelial cells. In oral lichen planus-activated fibroblasts, 10 μM GB1107 reduced the proliferation and migration capacity, decreased the expression of α-SMA, FAP, VEGF, and FGF2, and inhibited the tube-forming capacity and the expression of VEGFR2 phosphorylation and FAK in co-cultured human umbilical vein vascular endothelial cells. The upregulation of Galectin-3 expression in oral lichen planus is associated with angiogenesis, and the oral lichen planus-activated fibroblasts promote human umbilical vein vascular endothelial cells migration and tube-forming differentiation through VEGFR2/FAP activation by Galectin-3.

Arecoline promotes fibroblast activation and M2-macrophage polarization by up-regulating the expression of IL-4

ObjectiveTo determine the biological effects of arecoline on oral submucous fibrosis (OSF). DesignThe differential genes between OSF tissue and normal oral tissue were collected form GSE64216 dataset, analyzed by Gene Expression Omnibus (GEO) database. Real-time PCR and immunohistochemistry were used to analyze the expression of IL-4 gene and protein in oral tissue. Enzyme-linked immunosorbent assay (ELISA) was used to analyze the expression of exocrine IL-4 protein in human oral fibroblasts (HOF) pre-treated by arecoline. Cell Counting Kit-8 (CCK-8) and transwell assays were used to analyze the proliferation and migration of HOF cells, respectively. After IL-4 was knocked down by short hairpin (sh) plasmid, the proliferation and migration of HOF cells were detected. Flow cytometry was used to analyze the proportion of M2-macrophages. Real-time PCR and immunohistochemistry were used to verify the expression of biomarker proteins of macrophages in OSF tissues. ResultsThe expression of IL-4 gene and protein were both up-regulated in OSF tissue. Arecoline could enhance the expression of IL-4 gene and exocrine protein in HOF cells, and promote the proliferation and migration of HOF cells. While knockdown of IL-4 could inhibit arecoline-induced proliferation and migration in HOF cells. The results of flow cytometry showed that recombinant human IL-4 (rhIL-4) protein could increase the proportion of M2-macrophages. Similarly, the results of real-time PCR and immunohistochemistry showed the expression of ARG1 (Biomarker proteins of M2-macrophage) was up-regulated in OSF tissues. ConclusionArecoline promotes activation of fibroblasts and polarization of M2-macrophages by up-regulating the expression of IL-4.

Birefringence mapping of biological tissues based on polarization sensitive non-interferometric quantitative phase imaging technique

ObjectiveOral cancer is a leading cause of mortality globally, particularly affecting developing regions where oral hygiene is often overlooked. The optical properties of tissues are vital for diagnostics, with polarization imaging emerging as a label-free, contrast-enhancing technique widely employed in medical and scientific research over past few decades. Materials and methodsWe present a novel polarization sensitive quantitative phase imaging of biological tissues by incorporating the conventional polarization microscope and transport of intensity equation-based phase retrieval algorithm. This integration provides access to the birefringence mapping of biological tissues. The inherent optical anisotropy in biological tissues induces the polarization dependent refractive index variations which can provide the detailed insights into the birefringence characteristics of their extracellular constituents. Experimental investigations were conducted on both normal and cancerous oral tissue samples by recording a set of three polarization intensity images for each case with a step size of 2 μm. ResultsA noteworthy increment in birefringence quantification was observed in cancerous as compared to the normal tissues, attributed to the proliferation of abnormal cells during cancer progression.The mean birefringence values were calculated for both normal and cancerous tissues, revealing a significant increase in birefringence of cancerous tissues (2.1 ± 0.2) × 10−2 compared to normal tissues (0.8 ± 0.2) × 10−2. Data were collected from 8 patients in each group under identical experimental conditions. ConclusionThis polarization sensitive non-interferometric optical approach demonstrated effective discrimination between cancerous and normal tissues, with various parameters indicating elevated values in cancerous tissues.

Abstract 6992: Profilin 2 (PFN2) promotes the proliferation, migration, invasion and epithelial-to-mesenchymal transition of oral squamous cell carcinoma

Abstract Background:Oral squamous cell carcinoma (OSCC) stands as a prevalent malignancy worldwide. Profilin 2 (PFN2), an actin-binding protein regulating actin polymerization dynamics and associated with cell motility, has recently gained attention as a significant regulator in various cancers. However, the clinical significance and biological function of PFN2 in OSCC remain debatable. This study aims to elucidate the role of PFN2 in OSCC. Methods:We assessed PFN2 expression in OSCC and adjacent normal oral mucosa tissues through Q-RT-PCR and immunohistochemistry analysis. Associations between PFN2 expression, clinicopathological features, and OSCC prognosis were analyzed. Cell proliferation, invasion, and migration effects were examined via MTT and Transwell assays. Western blotting assessed the protein expression related to epithelial–mesenchymal transition (EMT). Additionally, we established OSCC xenografts to determine tumorigenicity in vivo using female Nude mice. Results:PFN2 RNA and protein expression were significantly increased in OSCC compared to normal oral mucosa. PFN2 expression positively correlated with lymphovascular invasion and cervical lymph node metastasis. MTT and Transwell assays demonstrated that PFN2 promoted OSCC cell proliferation, migration, invasion, and endothelial invasion. Downregulation of PFN2 induced an EMT phenotype, including an increase in the expression of the epithelial marker E-cadherin and a decrease in the expression of mesenchymal markers Vimentin, Snail, and Slug in OSCC cells in vitro. Similarly, OSCC cells with reduced PFN2 expression exhibited weaker tumorigenicity in vivo. Conclusions:Our findings unveil a novel role for PFN2 in promoting OSCC progression and metastasis, suggesting its potential as an early biomarker for high-risk populations. Targeting PFN2 may offer a promising therapeutic strategy for OSCC treatment. Citation Format: Chung Ji LIu, Li-Han Lin, Kuo-Wei Chang, Hui-Wen Cheng. Profilin 2 (PFN2) promotes the proliferation, migration, invasion and epithelial-to-mesenchymal transition of oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6992.

NCAPD2 promotes the malignant progression of oral squamous cell carcinoma via the Wnt/β-catenin pathway

ABSTRACT Oral squamous cell carcinoma (OSCC) is the most common type of oral cancer, with a poor prognosis, yet the underlying mechanism needs further exploration. Non-SMC condensin I complex subunit D2 (NCAPD2) is a widely expressed protein in OSCC, but its role in tumor development is unclear. This study aimed to explore NCAPD2 expression and its biological function in OSCC. NCAPD2 expression in OSCC cell lines and tissue specimens was analyzed using quantitative polymerase chain reaction, western blotting, and immunohistochemistry. Cancer cell growth was evaluated using cell proliferation, 5-Ethynyl-2’-deoxyuridine (EdU) staining, and colony formation assays. Cell migration was evaluated using wound healing and Transwell assays. Apoptosis was detected using flow cytometry. The influence of NCAPD2 on tumor growth in vivo was evaluated in a mouse xenograft model. NCAPD2 expression was significantly higher in OSCC than that in normal oral tissue. In vitro, the knockdown of NCAPD2 inhibited OSCC cell proliferation and promoted apoptosis. NCAPD2 depletion also significantly inhibited the migration of OSCC cells. Moreover, NCAPD2 overexpression induced inverse effects on OSCC cell phenotypes. In vivo, we demonstrated that downregulating NCAPD2 could inhibit the tumorigenicity of OSCC cells. Mechanically, OSCC regulation by NCAPD2 involved the Wnt/β-catenin signaling pathway. These results suggest NCAPD2 as a novel oncogene with an important role in OSCC development and a candidate therapeutic target for OSCC.

Prognostic significance of RKIP, TGM2, and CMTM4 expression in oral squamous cell carcinoma.

The expression of RKIP, TGM2, and CMTM4 in oral squamous cell carcinoma (OSCC) and normal oral tissues was detected and their correlations were analyzed. The relationships between RKIP, TGM2, and CMTM4 and the clinicopathological parameters and prognosis of patients were analyzed. Seventy cancerous and adjacent normal tissue samples were selected, recorded in the pathology department, and embedded in paraffin. Protein expression was detected by immunohistochemistry. Statistical software (SPSS 25.0, IBM Corporation) was used for the statistical analysis. The chi-squared (χ2) test was used to analyze the expression of RKIP, TGM2, and CMTM4 proteins and their clinicopathological features. Differences in RKIP, TGM2, and CMTM4 protein levels between OSCC and normal tissues were compared using a χ2 test. Survival analysis was performed using the Kaplan-Meier method, and differences between survival curves were determined using the log-rank test. The effects of RKIP, TGM2, and CMTM4 expression on patient prognosis were analyzed using a multivariate Cox proportional hazards regression model. P < .05 was considered statistically significant. The expression level of RKIP correlated with age and clinical stage (P < .05). TGM2 was associated with clinical stage and lymph node metastasis (P < .05). The expression of CMTM4 increased with a decrease in cancer differentiation. Kaplan-Meier survival analysis suggested that the positive expression of TGM2 and CMTM4 may predict poor prognosis in patients with OSCC. The multivariate Cox proportional hazards regression model suggested that TGM2 could be an independent prognostic factor for patients with OSCC. Combined expression of TGM2 and CMTM4 can be used as an indicator to evaluate the risk of metastasis and prognosis of OSCC.

Malignant Transformation of Normal Oral Tissue to Dysplasia and Early Oral Squamous Cell Carcinoma: An In Silico Transcriptomics Approach.

Background: Oral squamous cell carcinoma (OSCC) is a prevalent and aggressive form of head and neck cancer, often diagnosed at advanced stages. Elucidating the molecular mechanisms involved in the malignant transformation from normal oral tissue to oral preinvasive lesions (OPL) and primary OSCC could facilitate early diagnosis and improve therapeutic strategies. Methods: Differentially expressed genes (DEGs) were identified from the GSE30784 dataset by comparing normal oral tissue, oral dysplasia, and primary OSCC samples. Cross-validation was performed using an independent RNA-seq dataset, GSE186775. Protein-protein interaction (PPI) network analysis, gene ontology annotation, and pathway enrichment analysis were conducted on the common DEGs. Hub genes were identified, and their prognostic significance was evaluated using survival analysis. Transcription factor (TF) enrichment analysis, cross-validation, and immunohistochemistry analyses were also performed. Results: A total of 226 proteins and 677 interactions were identified in the PPI network, with 34 hub genes, including FN1, SERPINE1, PLAUR, THBS1, and ITGA6. Pathways such as "Formation of the cornified envelope," "Keratinization," and "Developmental biology" were enriched. Overexpression of SERPINE1, PLAUR, THBS1, and ITGA6 correlated with poor prognosis, while upregulation of CALML5 and SPINK5 was associated with favorable outcomes. NFIB emerged as the most significant TF-regulating hub genes. Immunohistochemistry validated ITGA6 overexpression in primary OSCC. Cross-validation using the RNA-seq dataset supported the involvement of critical genes in the malignant transformation process. Conclusion: This study identified vital genes, pathways, and prognostic markers involved in the malignant transformation from normal oral tissue to OPL and primary OSCC, providing insights for early diagnosis and targeted therapy development. Cross-validation with an independent RNA-seq dataset and immunohistochemistry reinforced the findings, supporting the robustness of the identified molecular signatures.

Linking inflammation and angiogenesis with fibrogenesis: Expression of FXIIIA, MMP-9, and VEGF in oral submucous fibrosis

ObjectivesInterplay of Factor XIIIa (FXIIIa), a transglutaminase, responsible for cross-linking of matrix proteins, Matrix Metalloproteinase-9 (MMP-9), a gelatinase, and Vascular Endothelial Growth Factor (VEGF), an angiogenic inducer, were studied in relation to fibrogenesis and disease progression in oral submucous fibrosis (OSMF). Material and methodsImmunohistochemical expression of markers was studied in 60 formalin-fixed paraffin-embedded tissue blocks of OSMF and 20 normal oral mucosal tissues. FXIIIa was studied quantitatively while MMP-9 and VEGF were assessed semi-quantitatively. Expression was compared with histopathological grades of OSMF. ResultsFXIIIa expression significantly increased in OSMF (p-value 0.000). However, expression decreased and cells became quiescent with increasing grades (p-value 0.000). MMP-9 (p-value epithelium 0.011, p-value connective tissue 0.000) and VEGF expression (p-value epithelium 0.000, connective tissue 0.000) increased in OSMF. A negative correlation between FXIIIa and MMP-9 (−0.653) in early grade (p-value of 0.021) and a positive correlation between FXIIIa and VEGF (0.595) (p-value of 0.032) was found in the moderate grade OSMF. Regression analysis showed a significant association (p<0.01) of FXIIIa in OSMF and with increasing grades of OSMF. ConclusionFXIIIa may play a crucial role in initiation of fibrosis in OSMF. MMP-9 may have a diverse role to play in OSMF as a regulator of fibrosis. VEGF may show an angio-fibrotic switch and contribute to fibrosis in OSMF. These cytokines may show altered function and can contribute to fibrosis and chronicity of disease due to changes in the microenvironment. Tissue stiffness in OSMF itself creates an environment that enhances the chronicity of the disease.

Correlation of PD-1 and PD-L1 expression in oral leukoplakia and oral squamous cell carcinoma: an immunohistochemical study

The programmed cell death protein (PD-1)/programmed cell death protein ligand (PD-L1) pathway and cytotoxic T lymphocyte antigen are the most important co-stimulatory molecules that play a key role in the negative regulation of T cells during carcinogenesis. We aimed to evaluate the immunohistochemical expression of PD-1 and PD-L1 in oral leukoplakia and squamous cell carcinoma compared with normal oral mucosa. Twenty-five cases of oral squamous cell carcinoma, oral leukoplakia and normal oral mucosa tissue specimens were immunohistochemically stained to assess PD-1 and PD-L1 expression. The PD-L1 positivity of subepithelial TAFs (p < 0.001) increased with increasing grades of oral leukoplakia. Pearson’s correlation indicated a high positive correlation between the PD-L1 labelling index of epithelial tumour cells and the PD-1 labelling index of tumour infiltrating lymphocytes (p value: 0.005) in OSCC. A high positive correlation was noted between the H-score of PD-L1 positive tumour epithelial cells and the H-score of PD-1 positive tumour infiltrating lymphocytes in OSCC (p value: 0.001). PD-L1 positivity increased in dysplastic epithelial cells from premalignant lesions to malignancy. The sub-epithelial PD-L1 positive TAFs were higher in oral leukoplakia compared to OSCC inferring that PD-L1 positivity in TAFs decreased with malignant transformation. The PD-1 positivity in TILs was higher in oral leukoplakia than in OSCC.

RCN1 deficiency inhibits oral squamous cell carcinoma progression and THP-1 macrophage M2 polarization

Reticulocalbin 1 (RCN1), a calcium-binding protein located in the endoplasmic reticulum (ER) lumen, contains six conserved regions. Its main functions include maintaining intracellular homeostasis and regulating cell proliferation and apoptosis, and it plays an important role in the development of various tumours. However, the exact function of RCN1 in oral squamous cell carcinoma (OSCC) is not fully understood. Therefore, the aim of this study was to investigate the effects of RCN1 on the biological behaviour of OSCC and the regulation of tumour-associated macrophage (TAM) polarization. The expression of RCN1 in OSCC and normal oral mucosa was evaluated through bioinformatics analysis and immunohistochemical staining. The growth, migration, and invasion of OSCC cells were observed after knockdown of RCN1 using CCK-8 and Transwell assays. Apoptosis was detected by flow cytometry. The effect of tumour cell-derived RCN1 on the polarization of THP-1 macrophages was investigated by establishing a coculture model of THP-1 macrophages and OSCC cells. Additionally, changes in the expression levels of relevant proteins were detected using Western blotting. The upregulation of RCN1 in tumour tissues compared to normal oral mucosal tissues is associated with a poor prognosis and can be utilized as a prognostic indicator for OSCC. Knockdown of RCN1 inhibited the proliferation, migration, and invasion of OSCC cells. Additionally, knockdown of RCN1 in Cal-27 and SCC-25 cells resulted in inhibition of the M2 polarization of THP-1 macrophages. RCN1 knockdown inhibits OSCC progression and M2 macrophage polarization. Targeting RCN1 may be a promising approach for OSCC treatment.

Evaluation of USP22 and Ki-67 expression in oral squamous cell carcinoma: An immunohistochemical study.

USP22 is a positive regulator in tumor growth, its depletion leads to cell cycle arrest at G1 phase. USP22 over expression was positively correlated with proteins involved in proliferation and negatively correlated with tumor suppressor protein tumor supprn. Ki-67 expression is associated with USP22 over expression in oral squamous cell carcinoma (OSCC) and also in cervical and prostate cancers. The aim of this study is to evaluate the expression of USP22 and Ki-67 in OSCC by using an immunohistochemical staining procedure. Immunohistochemistry was used to determine the expression of USP22 protein in 50 archival tissue blocks of histopathologically diagnosed OSCC and 15 normal oral mucosa tissue blocks. The histopathological correlation of USP22 with Ki-67 was done. Expression of USP22 and Ki-67 was seen in the nuclei of epithelial cells. Statistical analysis of the mean expression of USP22 in OSCC and normal tissue showed a significant difference (P = 0.000000119). A significant difference was also observed in Ki-67 between OSCC and normal tissue (P = 0.00000086). Correlation test showed a weak correlation (R = 0.19) between USP22 and Ki-67 expression of group 1. Similarly, a weak correlation (R = 0.51) was observed in group 2. A statistically significant difference in the expression of USP22 and Ki-67 was observed between normal mucosa and OSCC. It can be used in early diagnosis of OSCC but its use as a prognostic indicator is questionable and should be exemplified with a larger study sample.

Activation of oncogenic signaling kinase PAK1 by ionising radiation confers an aggressive phenotype in head and neck squamous cell carcinoma

Head and neck squamous cancers are very aggressive tumors often diagnosed in late stages with poor prognosis. HNSCCs are usually treated by a course of radiation (IR) therapy and followed by surgery. These treatment regimens fail to bring a complete response. Molecular signatures in tumors are attributed to this response and an improved understanding of the signaling events could offer new avenues for therapy. Here, we show that P21 activated kinase-1 (PAK1) - an oncogenic signaling serine/threonine kinase, is activated upon exposure to IR and this leads to an accelerated tumorigenic character in HNSCC cells. Our results show that PAK1 is highly expressed in HNSCC cell lines, as compared to normal buccal mucosa cells and when HNSCC cells were exposed to IR, they show activated PAK1 and an aggressive phenotype as determined by in vitro functional assays. PAK1 levels were elevated in HNSCC as compared to adjacent normal oral tissues and our results also show convincing evidence of activated PAK1 in patient tumor samples of post- IR treatment as compared to pre-IR treatment and is associated with poor survival. Pak1 Knockout (KO) clones in HNSCC cells showed that they were more sensitive to IR as compared to wild type (wt) cells. This altered sensitivity to IR was attributed to enhanced DNA damage response modulated by PAK1 in cells. Overall, our results suggest that PAK1 expression in HNSCC could be a critical determinant in IR therapy response and silencing PAK1 is likely to be a treatment modality to improve clinical outcomes.