Oral Squamous Cell Carcinoma Samples Research Articles

Aldehyde Dehydrogenase: An Off-Label Marker of Endothelial Activation in Oral Squamous Cell Carcinoma.

The vascular endothelial (VE) expression of aldehyde dehydrogenase (ALDH) 1/2 family in oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC) cases has not been studied so far. The aim of this study was to illustrate the "off-label" endothelial expression of cancer stem cell (CSC) biomarker, ALDH1/2, adjacent to oral potentially malignant and malignant lesions in order to shed some light on the mechanisms leading to oral carcinogenesis. Materials and methods: The expression of CSC protein-biomarker ALDH1/2 was detected through immunohistochemistry (IHC) in 30 paraffin-embedded samples of OL and 21 samples of OSCC compared to five samples of normal oral mucosa. Statistical analysis was done using SPSS, Pearson Chi-square, and Fischer's exact test. The significance level was set at 0.05 (p≤ 0.05). Results: In oral mucosal vessels, ALDH1/2 was not expressed. It was expressed significantly more in the vessels of OSCCs compared to the OLs (Fisher's exact test, p-value= 0,001). However, when endothelial expression of ALDH1/2 in the vasculature of OLs was compared with that of the normal oral mucosa, no significant change was noticed (Fisher's exact test, p-value=1.000). Discussion: The IHC VE expression of ALDH1/2 in OSCCvasculature but not in OL indicates a possible significantly stronger activation of endothelial cells during carcinogenesis, which could be an indicator of the role of inflammation in the development of field cancerization and of prognostic value for (vascular/lymphatic) metastasis.

A2b and A3 adenosine receptors may act as promotive and prognostic factors in oral squamous cell carcinoma

This study aimed to examine the expression and potential effects of adenosine receptors in oral squamous cell carcinoma (OSCC).Data on mRNA expression of adenosine receptors in OSCC samples were collected from The Cancer Genome Atlas database. Adenosine-regulated signaling pathways and biological processes were investigated via immune cell infiltration analysis, bioinformatics analysis, and immunohistochemistry.Overexpression of A2bR and A3R was significantly correlated with the prognosis of OSCC (P < 0.05). A3R expression in OSCC patients was significantly and positively correlated with the infiltration of six types of immune cells: B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and myeloid dendritic cells. A2bR expression weakly and negatively correlated with B-cell infiltration only. The expression of A2bR in OSCC was positively correlated with E-cadherin and PCNA, while the expression of A3R was positively correlated with that of cleaved caspase-3.Within the limitations of the study it seems that the overexpression of A2bR and A3R results in the poor prognosis of OSCC, suggesting that A2bR promotes cell proliferation in OSCC, while A3R may be involved in OSCC progression by regulating tumor cell apoptosis and immune microenvironment.

Expression of Epsin3 and its interaction with Notch signalling in oral epithelial dysplasia and oral squamous cell carcinoma.

Most oral squamous cell carcinoma patients present with late-stage disease. Early detection of the disease is considered to be the most effective way of improving patient outcomes. Several biomarkers have been identified as indicators of oral cancer development and progression; however, none have been translated into clinical practice. In this study, we have investigated the role of Epsin3, an endocytic adaptor protein, and Notch1, a transmembrane signalling protein, in oral carcinogenesis with a view to explore their potential as biomarkers. Oral cancer cell lines and a normal oral keratinocyte cell line were used together with tissue samples of normal oral mucosa (n = 21), oral epithelial dysplasia (n = 74) and early stage (Stages I and II) oral squamous cell carcinoma (n = 31). Immunocytochemical staining, immunoblotting and real-time quantitative polymerase chain reaction (PCR) were performed to assess protein as well as gene expression levels. The expression levels of Epsin3 and Notch1 mRNA and protein are variable across different oral squamous cell carcinoma derived cell lines. Epsin3 was upregulated in oral epithelial dysplasia and oral squamous cell carcinoma tissues compared with normal epithelium. Overexpression of Epsin3 resulted in a significant reduction of Notch1 expression in oral squamous cell carcinoma. Notch1 was generally downregulated in the dysplasia and oral squamous cell carcinoma samples. Epsin3 is upregulated in oral epithelial dysplasia and oral squamous cell carcinoma and has the potential to be used as a biomarker for oral epithelial dysplasia. Notch signalling is downregulated in oral squamous cell carcinoma, possibly through an Epsin3-induced de-activation pathway.

Identification of a Favorable Prognostic Subgroup in Oral Squamous Cell Carcinoma: Characterization of ITGB4/PD-L1high with CD8/PD-1high

Integrin β4 (ITGB4) is a member of the integrin family, which plays a crucial role in mediating cell adhesion to the extracellular matrix. Recent studies have demonstrated that ITGB4 is involved in tumorigenesis and metastasis during the development of cancer. However, the role of ITGB4 in oral squamous cell carcinoma (OSCC) remains unclear. A Multiplex immunohistochemistry (OPAL™, mIHC) assay was employed to stain ITGB4, ALDH1, PD-L1, cytokeratin (CK), CD8 and PD-1 in a human OSCC tissue microarray, containing 26 normal oral epithelium samples, 21 oral epithelium dysplasia samples and 76 OSCC samples. The expression pattern and clinicopathological characteristics of ITGB4 were analyzed and compared with those of PD-1, PD-L1, ALDH1 and CD8. The correlation between subgroups of tumor cells, including ITGB4+PD-L1+ and ITGB4+ALDH1+, and subgroups of T cells, including CD8+ and CD8+PD-1+, was evaluated using two-tailed Pearson’s statistics. A Kaplan–Meier curve was built, and a log-rank test was performed to analyze the survival rate of different subgroups. The mIHC staining results show that ITGB4 was mostly expressed in the tumor cells, with a significant increase in the OSCC specimens compared with normal oral epithelium and oral epithelium dysplasia. The paired analysis, conducted between the OSCC tumor tissue and normal paracancer mucosa, confirmed the results. The study further revealed that ITGB4+PD-L1+ cancer cells, but not ITGB4+ALDH1+ cancer cells, were significantly associated with the infiltration of CD8+ T cells (positivity p = 0.005, positive number p = 0.03). Additionally, ITGB4+PD-L1+ tumor cells were positively correlated with CD8+PD-1+ T cells (positivity p = 0.02, positive number p = 0.03). Most intriguingly, the subgroup of ITGB4/PD-L1high with CD8/PD-1high displayed the best prognosis compared with the other considered subgroups. The results show that the expression of ITGB4 was increased in OSCC compared with normal oral mucosa. Furthermore, a specific subgroup with high levels of expression of ITGB4/PD-L1 and CD8/PD-1 was found to have a relatively better prognosis compared with the other subgroups. Ultimately, this study sheds light on the potential role of ITGB4 in OSCC and provides a basis for further investigation.

Evaluating the expression of microRNA-15a-5p and YAP1 gene in oral squamous cell carcinoma in comparison with normal tissue: A cross-sectional study.

MicroRNAs (miRNAs) play crucial regulatory roles in cancer progression, including oral cancer (OC). However, the biological mechanisms of miRNA-15a-5p in OC remain unclear. This study aimed to evaluate the expression of miRNA-15a-5p and the YAP1 gene in OC. A total of 22 clinically and histologically confirmed oral squamous cell carcinoma (OSCC) patients were recruited, and their tissues were stored in a stabilizing solution. Later, RT-PCR was performed to evaluate miRNA-15a-5p and the targeting gene YAP1. The results of OSCC samples were compared with unpaired normal tissues. The normality tests, Kolmogorov-Smirnov and Shapiro-Wilk, revealed a normal distribution. Inferential statistics were performed using an independent sample t-test/unpaired t-test among the study intervals to compare the expression of miR-15a and YAP1. SPSS (IBM SPSS Statistics for Windows, Version 26.0, Armonk, NY: IBM Corp. Released 2019) was used to analyse the data. The significance level was set at 5% (α = 0.05), and a p-value <0.05 was considered statistically significant. The expression of miRNA-15a-5p was lower in OSCC than in normal tissue, whereas the opposite was observed for YAP1 levels. In conclusion, this study demonstrated that miRNA-15a-5p was downregulated and YAP1 was overexpressed, which had a statistically significant difference between the normal and OSCC groups. Therefore, miRNA-15a-5p may serve as a novel biomarker to better understand the pathology and as a potential target in OSCC therapy.

Overexpression of heat-shock protein 47 impacts survival of patients with oral squamous cell carcinoma.

The expression of heat-shock protein 47 (HSP47) has been linked to collagen synthesis control and implicated in fibrotic disorders, but more recent studies have demonstrated its role in solid tumors. In this study, we explored the prognostic impact of HSP47 in oral squamous cell carcinomas (OSCC) and determined the in vitro effects of its loss-of-function on viability, proliferation, migration, invasion, and resistance to cisplatin of OSCC cells. The HSP47 expression in tumor samples was assessed by immunohistochemistry in two independent cohorts totaling 339 patients with OSCC, and protein levels were associated with clinicopathological features and survival outcomes. The OSCC cell lines HSC3 and SCC9 were transduced with lentivirus expressing short hairpin RNA to stably silence HSP47 and used in assays to measure cellular viability, proliferation, migration, and invasion. HSP47 was overexpressed in OSCC samples, and its overexpression was significantly and independently associated with poor disease-specific survival and shortened disease-free survival in both OSCC cohorts. The knockdown of HSP47 showed no effects on cell viability or cisplatin sensitivity, but impaired significantly proliferation, migration, and invasion of OSCC cells, with stronger effects on SCC9 cells. Our results show a significant prognostic impact of HSP47 overexpression in OSCC and reveal that HSP47 inhibition impairs the proliferation, migration, and invasion of OSCC cells. HSP47 may represent a potential therapeutic target for OSCC.

CDH4 inhibits ferroptosis in oral squamous cell carcinoma cells

BackgroundThe cadherin-4 gene (CDH4), a member of the cadherin family genes, encodes R-cadherin (R-cad); however, the function of this gene in different types of cancer remains controversial. The function of CDH4 in OSCC (oral squamous cell carcinoma) is unknown.Materials and methodsWe use the Cancer Genome Atlas (TCGA) database to find the expression of CDH4 in OSCC is more than normal tissue. Our tissue samples also confirmed that CDH4 gene was highly expressed in OSCC. The related cell function assay detected that CDH4 promotes the ability of cell proliferation, migration, self-renewal and invasion. Cell staining experiment confirmed that the change of CDH4 expression would change the cell mortality. The western blot of GPX4 (glutathione-dependent peroxidase-4), GSH (reduced glutathione) test assay and MDA(Malondialdehyde) test assay show that the expression of CDH4 may resist the sensitivity of ferropotosis in OSCC.ResultsCDH4 was upregulated in OSCC samples and was correlation with poor survival of patients. High expression of CDH4 effectively promotes the proliferation, mobility of OSCC cells and reduce the sensitivity of OSCC cells to ferroptosis. CDH4 is positively correlated with EMT pathway genes, negatively correlated with fatty acid metabolism pathway genes and peroxisome pathway genes, and positively correlated with ferroptosis suppressor genes in OSCC.ConclusionsThese results indicate that CDH4 may play a positive role in tumor progression and resistance ferroptosis and may be a potential therapeutic target for OSCC.

Histone lysine methyltransferase SMYD3 promotes oral squamous cell carcinoma tumorigenesis via H3K4me3-mediated HMGA2 transcription

BackgroundEpigenetic dysregulation is essential to the tumorigenesis of oral squamous cell carcinoma (OSCC). SET and MYND domain-containing protein 3 (SMYD3), a histone lysine methyltransferase, is implicated in gene transcription regulation and tumor development. However, the roles of SMYD3 in OSCC initiation are not fully understood. The present study investigated the biological functions and mechanisms involved in the SMYD3-mediated tumorigenesis of OSCC utilizing bioinformatic approaches and validation assays with the aim of informing the development of targeted therapies for OSCC.Results429 chromatin regulators were screened by a machine learning approach and aberrant expression of SMYD3 was found to be closely associated with OSCC formation and poor prognosis. Data profiling of single-cell and tissue demonstrated that upregulated SMYD3 significantly correlated with aggressive clinicopathological features of OSCC. Alterations in copy number and DNA methylation patterns may contribute to SMYD3 overexpression. Functional experimental results suggested that SMYD3 enhanced cancer cell stemness and proliferation in vitro and tumor growth in vivo. SMYD3 was observed to bind to the High Mobility Group AT-Hook 2 (HMGA2) promoter and elevated tri-methylation of histone H3 lysine 4 at the corresponding site was responsible for transactivating HMGA2. SMYD3 also was positively linked to HMGA2 expression in OSCC samples. Furthermore, treatment with the SMYD3 chemical inhibitor BCI-121 exerted anti-tumor effects.ConclusionsHistone methyltransferase activity and transcription-potentiating function of SMYD3 were found to be essential for tumorigenesis and the SMYD3–HMGA2 is a potential therapeutic target in OSCC.

Exosomes derived from oral squamous cell carcinoma tissue accelerate diabetic wound healing.

It is a widespread and difficult problem that refractory diabetic wounds have a poor local environment and prolonged inflammatory irritation. Tumor cell-derived exosomes play an important role in the development of tumors, as they can promote tumor cell proliferation, migration, and invasion and enhance tumor cell activity. However, tumor tissue-derived exosomes have been less studied, and it is unclear how they affect wound healing. In this study, we extracted tissue-derived exosomes from human oral squamous carcinoma and paracancerous tissue by ultracentrifugation, size exclusion chromatography and ultrafiltration and performed exosome characterization. In vitro, the oral squamous cell carcinoma tissue-derived exosomes (OSCC Ti-Exos) promoted the proliferation and migration of endothelial cells, keratinocytes, and fibroblasts. In addition, in vivo experiments showed that the OSCC Ti-Exos accelerated the healing of diabetic wounds and were safe in mice. In contrast, there was no promoting effect of paracancerous tissue-derived exosomes either in vivo or in vitro. In conclusion, OSCC Ti-Exos promoted the healing of diabetic wounds, demonstrated preliminary biosafety in mice and have promise as therapeutic applications.

Altered oral microbiome in Sudanese Toombak smokeless tobacco users carries a newly emerging risk of squamous cell carcinoma development and progression

There are an estimated 6–10 million smokeless tobacco (Toombak) users in Sudan, the majority being males. Toombak is known to be a carcinogenic product that is likely to modify the oral microbiome spatiality into a high-risk potential for the development and progression of oral cancer, but previous studies are lacking in this field. Here, we endeavour for the first time the exploration of the oral microbiome in key mucosal areas of the oral cavity and assess the microbiome variations in premalignant and oral squamous cell carcinoma (OSCC) samples from both users and non-users of Toombak. 16S rRNA sequencing was performed on DNA obtained from pooled saliva, oral mucosa and supragingival plaque from 78 Sudanese users and non-users of Toombak, aged between 20 and 70 years. In 32 of the pooled saliva samples, the mycobiome (fungal) environment was analysed through ITS sequencing. Then, 46 formalin-fixed paraffin-embedded samples of premalignant and OSCC samples were collected, and their associated microbiomes sequenced. The oral Sudanese microbiome was found to be enriched in Streptococcaceae, but Staphylococcaceae were significantly more abundant amongst Toombak users. Genera enriched in the oral cavity of Toombak users included Corynebacterium_1 and Cardiobacterium while in non-users, Prevotella, Lactobacillus and Bifidobacterium were prominent. Aspergillus was the most abundant fungus in the mouths of Toombak users with a marked loss of Candida. The genus Corynebacterium_1 was abundant in the buccal, floor of the mouth and saliva microbiomes as well as in oral cancer samples from Toombak users indicating a possible role for this genus in the early stages of oral cancer development. An oral cancer microbiome that favours poor survival and metastasis in those who use Toombak also emerged that includes the genera Stenotrophomonas and Schlegelella. Those utilising Toombak carry an altered oral microbiome that may be an additional risk factor for this products carcinogenicity to the oral structures. These significant microbiome modulations are a newly emerging key driving factor in oral cancer development and progression in Toombak users while it is also shown that Toombak users carry an oral cancer microbiome that may increase the potential for a poorer prognosis.

Characterization of the m6A regulator-mediated methylation modification patterns in oral squamous cell carcinoma

N6-methyladenosine (m6A) is a form of posttranscriptional modification that plays important roles in cancer including oral squamous cell carcinoma (OSCC). Most studies to date have focused on a limited number of regulators and oncogenic pathways, thus failing to provide comprehensive insight into the dynamic effects of m6A modification. In addition, the role of m6A modification in shaping immune cell infiltration in OSCC has yet to be clarified. This study was designed to assess m6A modification dynamics in OSCC and to understand how such modifications influence clinical immunotherapeutic treatment outcomes. m6A modification patterns linked with 23 m6A regulators were analyzed in 437 OSCC patients from TCGA and GEO cohorts. These patterns were then quantified through m6A score based on algorithms derived from a principal component analysis (PCA) approach. The m6A modification patterns of OSCC samples were grouped into two clusters based on the m6A regulators expression, and immune cell infiltration was linked with the 5-year survival outcomes of patients in these clusters. 1575 genes associated with OSCC patient prognosis were identified and used to re-cluster these samples into two groups. Patients in clusters exhibiting higher levels of m6A regulator expression exhibited poorer overall survival (OS), whereas patients with high m6A scores survived for longer (p < 0.001). The overall mortality rates in the groups of patients with low and high m6A scores were 55% and 40%, respectively, and the m6A score distributions in clusters of patients grouped by m6A modification patterns and gene expression further supported the link between a high m6A score and better prognostic outcomes. Immunophenoscore (IPS) values for patients in different m6A score groups suggested that the use of PD-1-specific antibodies or CTLA-4 inhibitors alone or in combination would yield superior treatment outcomes in patients in the high-m6A score group relative to the low-m6A score group. m6A modification patterns are relevant to heterogeneity in OSCC. Detailed analyses of m6A modification patterns may thus offer novel insight regarding immune cell infiltration within the OSCC tumor microenvironment, guiding novel efforts to provide patients with more effective immunotherapeutic interventions.

Fibroblast growth factor receptors 1 and 4 combined with lymph node metastasis predicts poor prognosis in oral cancer.

The fibroblast growth factor receptor (FGFR) members including FGFR1-4 have been identified as promising novel therapeutic targets and prognostic markers in multiple solid tumors. However, the predictive role of the expression of FGFR proteins in oral squamous cell carcinoma (OSCC) requires further exploration. Immunohistochemical evaluation of FGFR1-4 was performed on 161 paired OSCC samples. The associations of FGFRs with clinicopathologic and prognostic parameters were analyzed. To further assess the contribution of FGFRs to OSCC proliferation, cell lines, and one PDX model was utilized to examine the anti-tumor effect of the pan-FGFR inhibitor AZD4547. All FGFR members were found to be overexpressed in OSCC tumors when compared to normal tissues, and their expression was significantly associated with poor overall survival and disease-free survival. Multivariate Cox regression analysis revealed high expression of FGFR1 (p = 0.014) and FGFR4 (p = 0.009) were independent prognostic factors and co-overexpression of FGFR1 and FGFR4 with lymph node metastasis increased HR for death (p = 0.02). The pan-FGFR inhibitor AZD4547 showed anti-tumor activity in cell lines and in a patient-derived xenograft of OSCC. This study highlights the co-overexpression of FGFR1 and FGFR4 as a significantly poor prognosis indicator in OSCC when combined with lymph node metastasis.

Identification of novel prognostic indicators for oral squamous cell carcinoma based on proteomics and metabolomics

BackgroundThe low 5-year survival rate of oral squamous cell carcinoma (OSCC) suggests that new prognostic indicators need to be identified to aid the clinical management of patients. MethodsSaliva samples from OSCC patients and healthy controls were collected for proteomic and metabolomic sequencing. Gene expressed profiling was downloaded from TCGA and GEO databases. After the differential analysis, proteins with a significant impact on the prognosis of OSCC patients were screened. Correlation analysis was performed with metabolites and core proteins were identified. Cox regression analysis was utilized to stratify OSCC samples based on core proteins. The prognostic predictive ability of the core protein was then evaluated. Differences in infiltration of immune cells between the different strata were identified. ResultsThere were 678 differentially expressed proteins (DEPs), 94 intersected DEPs among them by intersecting with differentially expressed genes in TCGA and GSE30784 dataset. Seven core proteins were identified that significantly affected OSCC patient survival and strongly correlated with differential metabolites (R2 > 0.8). The samples were divided into high- and low-risk groups according to median risk score. The risk score and core proteins were well prognostic factor in OSCC patients. Genes in high-risk group were enriched in Notch signaling pathway, epithelial mesenchymal transition (EMT), and angiogenesis. Core proteins were strongly associated with the immune status of OSCC patients. ConclusionsThe results established a 7-protein signatures with the hope of early detection and the capacity for risk assessment of OSCC patient prognosis. Further providing more potential targets for the treatment of OSCC.

The participation of tumor residing pericytes in oral squamous cell carcinoma

Pericytes are perivascular cells related to vessel structure and angiogenesis that can interact with neoplastic cells, interfering with cancer progression and outcomes. This study focused on the characterization of pericytes in oral squamous cell carcinoma (OSCC) using clinical samples and a transgenic mouse model of oral carcinogenesis. Nestin-/NG2+ (type-1) and nestin+/NG2+ (type-2) pericytes were analyzed by direct fluorescence after induction of oral carcinogenesis (4-nitroquinoline-1-oxide). Gene expression of neuron glial antigen-2 (NG2), platelet-derived growth factor receptor beta (PDGFR-β), and cluster of differentiation 31 (CD31) was examined in human OSCC tissues. The protein expression of von Willebrand factor and NG2 was assessed in oral leukoplakia (i.e., oral potentially malignant disorders) and OSCC samples. Additionally, clinicopathological aspects and survival data were correlated and validated by bioinformatics using The Cancer Genome Atlas (TCGA). Induction of carcinogenesis in mice produced an increase in both NG2+ pericyte subsets. In human OSCC, advanced-stage tumors showed a significant reduction in CD31 mRNA and von Willebrand factor-positive vessels. Low PDGFR-β expression was related to a shorter disease-free survival time, while NG2 mRNA overexpression was associated with a reduction in overall survival, consistent with the TCGA data. Herein, oral carcinogenesis resulted in an increase in NG2+ pericytes, which negatively affected survival outcomes.

Abstract 619: TP53 gain-of-function mutation modulates the immunosuppressive microenvironment in non-HPV associated oral squamous cell carcinoma

Abstract Background: TP53 is the most common mutated gene in head and neck cancer, including oral squamous cell carcinoma (OSCC), a lethal disease with limited therapeutic options. In OSCC, for example, TP53 R175H is a hot-spot, high-risk, functional mutation that inhibits the function of WT p53, however these correlations and functional roles of high-risk p53 mutations had not been experimentally addressed using syngeneic oral tumor models. To understand the functional role of TP53 mutations in modulating the tumor immune microenvironment (TIME) and response to immunotherapy, we used a syngeneic oral tumor model (ROC3 cell) derived from a genetically engineered mouse model expressing the p53 R172H (human, R175H). Methods: Orthotopic tongue injections were performed to (i) characterize tumor growth rate (ii) the TIME landscape by flow cytometry and opal multiplex immunofluorescence (mIF); and (iii) immunotherapy drug studies. We inactivated mutant p53 expression by shRNA and CRISPR Cas9, profiled gene expression changes by HTG EdgeSeq array, and validated these findings by qPCR, Western blot and immunochemistry. Tumor response was achieved by anti-PD1 and STING agonist therapies. Patients’ gene expression analyzed by CIBERSORT and TIME landscape performed by mIF. The spatial distribution of TIME was evaluated by Visiopharm software and UMAP. Results: We demonstrated that disruption of mutant p53R172H in ROC3 cells altered cytokine and chemokine expression levels and consequently affected the maturation and infiltration of different immune subsets, including cytotoxic CD8+ T cells, helper CD4+ T cells, Tregs, G-MDSCs, tumor-associated macrophages and neutrophils in ROC3 tumors. In addition, our spatial TIME analysis revealed that p53R172H mutation promoted the infiltration of Tregs, M2 macrophages, and exhausted CD8+ T cells, and excluded active CD8+ and CD11+ cells in both the tumor cores and on the tumor edges. Interestingly, it seems that p53R172H also regulates the spatial distribution of immunocyte populations, and their distribution between central and peripheral intratumoral locations. Moreover, we found that p53R172H mutation not only disrupted the phosphorylation of STING pathway which supported the recently reported finding, but also decreased the absolute amount of STING protein. Furthermore, we detected that p53-mutated tumor-infiltrating CD8+ and CD4+ T cells expressed programmed cell death protein 1 (PD-1), and that these tumors responded to anti-PD-1 and STING agonist therapy. Furthermore, Human OSCC samples also revealed associations between immune cell populations and the TP53R175H mutation, which paralleled the findings from our ROC3 tumor model. Conclusions: Our findings collectively demonstrate that the mutant TP53 GOF mutation modulates the tumor immune landscape and is associated with reduced overall survival in patients with OSCC. Citation Format: Yewen Shi, Xiaoyong Ren, Shaolong Cao, Tongxin Xie, Xi Chen, Chieko Michikawa, Abdullah A. Osman, Andrew G. Sikora, Jeffrey N. Myers, Roberto Rangel. TP53 gain-of-function mutation modulates the immunosuppressive microenvironment in non-HPV associated oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 619.

Pathogenetic model of survivin-dependent molecular signalling pathways in tumorigenesis of oral cancer and precursor lesions.

p53 tumour suppressor gene limits unchecked cellular growth in response to DNA damage, by causing G1 arrest and the activation of apoptosis. Inhibitors of apoptosis include survivin which acts by inhibition of caspases. Survivin has a significant role as a cell cycle modulator and is only minimally present in mature tissues. Aberrant expression of p53 and survivin has been evaluated in various carcinomas. Thus, the objective of this research was to elucidate the co-expression of p53 and survivin in tissue samples of Oral Potentially Malignant Disorders (OPMDs) and Oral Squamous Cell Carcinoma (OSCCs). Thirty tissue samples of OPMDs and 30 tissue samples of OSCCs taken from department archives were used in the study. Expression of p53 and survivin was analyzed in the study groups by the help of immunohistochemistry. Also, co-expression of both the markers was evaluated. The expression of p53 and survivin in the oral epithelium of patients with OSCCs was significantly higher than that in patients with OPMDs (P value ≤0.05). Our results provide insights into the altered survivin and p53 co-expression with significant immunoexpression within the study groups. Therefore, survivin and p53 could be better markers for identifying cell proliferation and apoptotic pathway. Also, malignant transformation rate of OPMD increases with increased expression of these markers.

ETS1 regulates NDRG1 to promote the proliferation, migration, and invasion of OSCC.

The aim of this study was to investigate the molecular mechanism by which the transcription factor ETS1 regulates N-myc downstream regulatory gene 1 (NDRG1) to provide a new theoretical basis for the study of oral squamous cell carcinoma (OSCC). In this study, eight human OSCC and paraneoplastic samples were collected. The expressions of NDRG1, ETS1, and Ki67 were detected by immunohistochemistry; apoptosis was detected by tdt-mediated dUTP notched end labeling; cell migration and invasion were detected by Transwell; quantitative real-time PCR was performed to detect the expression of NDRG1; RNA-binding protein immunoprecipitation (RIP) assays detected NDRG1 expression; immunofluorescence assays detected ETS1 expression. NDRG1 and ETS1 expression was significantly upregulated in cancer tissues and CAL-27 and SCC-6 cells. Knockdown of NDRG1 and ETS1 inhibited cell proliferation, migration, invasion, cloning, and EMT while promoting apoptosis and inhibited tumor development; ETS1 positively regulated NDRG1 expression; Finally, overexpression of NDRG1 in vivo and in vitro reversed the effect of ETS1 knockdown on CAL-27 and SCC-6 cells. ETS1 positively regulates the expression of NDRG1 and promotes OSCC. Therefore, ETS1 may serve as a new target for the clinical diagnosis and treatment of OSCC.

Cholesterol Is a Regulator of CAV1 Localization and Cell Migration in Oral Squamous Cell Carcinoma.

Cholesterol plays an important role in cancer progression, as it is utilized in membrane biogenesis and cell signaling. Cholesterol-lowering drugs have exhibited tumor-suppressive effects in oral squamous cell carcinoma (OSCC), suggesting that cholesterol is also essential in OSCC pathogenesis. However, the direct effects of cholesterol on OSCC cells remain unclear. Here, we investigated the role of cholesterol in OSCC with respect to caveolin-1 (CAV1), a cholesterol-binding protein involved in intracellular cholesterol transport. Cholesterol levels in OSCC cell lines were depleted using methyl-β-cyclodextrin and increased using the methyl-β-cyclodextrin-cholesterol complex. Functional analysis was performed using timelapse imaging, and CAV1 expression in cholesterol-manipulated cells was investigated using immunofluorescence and immunoblotting assays. CAV1 immunohistochemistry was performed on surgical OSCC samples. We observed that cholesterol addition induced polarized cell morphology, along with CAV1 localization at the trailing edge, and promoted cell migration. Moreover, CAV1 was upregulated in the lipid rafts and formed aggregates in the plasma membrane in cholesterol-added cells. High membranous CAV1 expression in tissue specimens was associated with OSCC recurrence. Therefore, cholesterol promotes the migration of OSCC cells by regulating cell polarity and CAV1 localization to the lipid raft. Furthermore, membranous CAV1 expression is a potential prognostic marker for OSCC patients.

Whole genome DNA methylation and mutational profiles identify novel changes in proliferative verrucous leukoplakia

Proliferative verrucous leukoplakia (PVL) is a rare form of oral leukoplakia with a relatively high transformation rate resulting in oral squamous cell carcinoma (OSCC). Molecular analysis of PVL at the genome level is limited and has only identified molecular similarities between PVL and OSCC. However, the clinical profile of PVL suggests that molecular differences may be more important. Whole exome sequencing of 5 PVL-associated OSCC (PVL-OSCC) and paired blood samples was used to identify somatic mutations common to the tumors. Whole methylome analysis of samples from 4 PVL-associated OSCC and 3 OSCC of non-PVL origin samples was conducted to explore differential methylation. In contrast to conventional OSCC, PVL-associated OSCC showed infrequent TP53 mutation and altered spectra of PIK3CA and NOTCH1 mutations. Unsupervised hierarchical clustering identified 63 probes that discriminated between PVL-associated OSCC and OSCC of non-PVL origin. Differences in methylation were most significant for divalent metal ion transport, particularly calcium movement. Specific differences in mutation and methylation profiles between PVL-derived OSCC and OSCC of non-PVL origin suggest differences in their transformation pathways. Further studies of early PVL lesions may identify markers of transformation that are also applicable to more common oral premalignant disorders such as oral epithelial dysplasia.

Point-of-care Analysis for Non-invasive Diagnosis of Oral cancer (PANDORA): A technology-development proof of concept diagnostic accuracy study of dielectrophoresis in patients with oral squamous cell carcinoma and dysplasia.

Delays in the identification and referral of oral cancer remain frequent. An accurate and non-invasive diagnostic test to be performed in primary care may help identifying oral cancer at an early stage and reduce mortality. Point-of-care Analysis for Non-invasive Diagnosis of Oral cancer (PANDORA) was a proof-of-concept prospective diagnostic accuracy study aimed at advancing the development of a dielectrophoresis-based diagnostic platform for oral squamous cell carcinoma (OSCC) and epithelial dysplasia (OED) using a novel automated DEPtech 3DEP analyser. The aim of PANDORA was to identify the set-up of the DEPtech 3DEP analyser associated with the highest diagnostic accuracy in identifying OSCC and OED from non-invasive brush biopsy samples, as compared to the gold standard test (histopathology). Measures of accuracy included sensitivity, specificity, positive and negative predictive value. Brush biopsies were collected from individuals with histologically proven OSCC and OED, histologically proven benign mucosal disease, and healthy mucosa (standard test), and analysed via dielectrophoresis (index test). 40 individuals with OSCC/OED and 79 with benign oral mucosal disease/healthy mucosa were recruited. Sensitivity and specificity of the index test was 86.8% (95% confidence interval [CI], 71.9%-95.6%) and 83.6% (95% CI, 73.0%-91.2%). Analysing OSCC samples separately led to higher diagnostic accuracy, with 92.0% (95% CI, 74.0%-99.0%) sensitivity and 94.5% (95% CI, 86.6%-98.5%) specificity. The DEPtech 3DEP analyser has the potential to identify OSCC and OED with notable diagnostic accuracy and warrants further investigation as a potential triage test in the primary care setting for patients who may need to progress along the diagnostic pathway and be offered a surgical biopsy.