Pathogenesis Of Oral Squamous Cell Carcinoma Research Articles

Co-upregulation of miR-31 and its host gene lncRNA MIR31HG in oral squamous cell carcinoma

Background/purposeSeveral long non-coding RNAs (lncRNAs) harbor miRNA in their genome. MIR31HG harbors miR-31 in its intron and it is speculated that they are co-expressed in tumors. This study addressed whether frequent miR-31 and MIR31HG co-upregulation occurred in oral squamous cell carcinoma (OSCC) and its clinical implications. Materials and methodsMicroarray was performed to retrieve dis-regulated lncRNAs from tissue sample. The ectopic gene expression was carried out to specify the phenotypic influences of selected lncRNA screened from bioinformatic algorithms. The expression of miR-31 and MIR31HG in tissues or scrapped samples was analyzed using qRT-PCR. The implications of gene expression as related to metastasis or survival were further dissected. ResultsMicroarray identified disrupted transcripts including MIR31HG and other 152 lncRNAs aberrantly expressed in OSCC tissues. In silico algorithms annotated an eminent involvement of aberrant transcripts in the regulation of cell cycle, extracellular modulation, adhesion, and wound healing. The enhancement of proliferation, wound healing, invasion and anchorage-independent colony formation mediated by MIR31HG was ascertained by ectopic expression in OECM1 cells. Besides, co-upregulation of miR-31 and MIR31HG was conspicuous in OSCC tissues. High expression of miR-31 and MIR31HG designated a trend of worse OSCC prognosis. Interestingly, high MIR31HG expression defined a very poor survival in stage IV diseases. By contrast, high miR-31 expression predicted nodal metastasis in stage I–III diseases. ConclusionAssessment of miR-31 and MIR31HG expression in OSCC may enable the prognostic prediction. The candidate lncRNAs isolated from this work can be further validated as crucial factors contributing to OSCC pathogenesis.

Molecular Pathogenesis of the Coronin Family: CORO2A Facilitates Migration and Invasion Abilities in Oral Squamous Cell Carcinoma.

In humans, the coronin family is composed of seven proteins containing WD-repeat domains that regulate actin-based cellular processes. Some members of the coronin family are closely associated with cancer cell migration and invasion. The Cancer Genome Atlas (TCGA) analysis revealed that CORO1C, CORO2A, and CORO7 were significantly upregulated in oral squamous cell carcinoma (OSCC) tissues (p < 0.05). Moreover, the high expression of CORO2A was significantly predictive of the 5-year survival rate of patients with OSCC (p = 0.0203). Overexpression of CORO2A was detected in OSCC clinical specimens by immunostaining. siRNA-mediated knockdown of CORO2A suppressed cancer cell migration and invasion abilities. Furthermore, we investigated the involvement of microRNAs (miRNAs) in the molecular mechanism underlying CORO2A overexpression in OSCC cells. TCGA analysis confirmed that tumor-suppressive miR-125b-5p and miR-140-5p were significantly downregulated in OSCC tissues. Notably, these miRNAs bound directly to the 3′-UTR of CORO2A and controlled CORO2A expression in OSCC cells. In summary, we found that aberrant expression of CORO2A facilitates the malignant transformation of OSCC cells, and that downregulation of tumor-suppressive miRNAs is involved in CORO2A overexpression. Elucidation of the interaction between genes and miRNAs will help reveal the molecular pathogenesis of OSCC.

INVESTIGATION OF SALIVARY miR-9, miR-34a ve miR-196a LEVELS IN FANCONI ANEMIA AND ORAL SQUAMOUS CELL CARCINOMA PATIENTS

Fanconi anemia (FA) is a rare bone marrow deficiency syndrome due to the DNA repair gene mutations, and Oral Squamous Cell Carcinoma (OSCC) is seen more frequently in FA patients than in the general population. The dysregulation of PI3K and Wnt signaling has been implicated in OSCC pathogenesis and abnormal expressions of miRNAs (a class of non-coding small regulatory RNAs) associated with these signaling pathways has been reported in OSHK patients. Salivary miRNAs are valuable biomarker candidates for OSCC development and prognosis. In this study, salivary levels of miR-9, miR-34a and miR-196a miRNAs related to PI3K and Wnt signaling pathways were examined in OSCC and FA patients and compared with the healthy control group. Saliva samples were collected from 89 subjects including 25 OSCC patients, 24 FA patients and 40 healthy controls. Total RNA was isolated using Quick-RNA Miniprep Kit (Zymo Research) due to the kit instructions. cDNA was generated with miRCURY LNA miRNA PCR Assay (Qiagen, Hilden, Germany) and Quantitative real-time PCR was performed with miRCURY LNA SYBR Green PCR Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. For the normalization of the expression levels of each miRNA, the mean expression U6 SnRNA was used as reference. The ΔΔCt value and the normalized miR-9, miR-34a and miR-196a salivary levels were calculated with Livak Method. Our results showed that miR-9 and miR-34a levels in OSCC patients were significantly lower compared to healthy control groups (p= 0,01 and p= 0,012), and there was no significant difference in miR-196a levels (p&gt; 0,05). In FA patients, miR-9 and miR-34 levels were lower than in control groups, likewise the OSCC patients (p =0,017 and p =0,014). There was no significant difference between miR-9, miR-34a, and miR-196a levels of FA patients and OSCC patients (p &gt;0.05). According to our results, low levels of miR-9 and miR-34a in saliva are biomarker candidates that may be important for OSCC development. In FA patients, close follow-up of the levels of miR-9 and miR-34 would be appropriate considering OSCC development. Further studies are needed to confirm the potential of miR-9 and miR-34a as biomarkers for OSCC.

P-element Induced WImpy protein-like RNA-mediated gene silencing 2 regulates tumor cell progression, apoptosis, and metastasis in oral squamous cell carcinoma.

ObjectiveP-element Induced WImpy protein-like RNA-mediated gene silencing 2 (PIWIL2) is a reported oncogene strongly associated with tumorigenesis and cancer progression. However, the potential function of PIWIL2 in oral cancer is still largely unclear.MethodsIn this study, we investigated the clinical significance of PIWIL2 expression in human oral squamous cell carcinoma (OSCC) cell lines and tissues. We also examined its function in OSCC pathogenesis by knocking down PIWIL2 expression with short hairpin RNAs, followed by phenotypic experiments focused on cell migration, invasion, proliferation, and apoptosis rates.ResultsWe found that PIWIL2 was overexpressed in OSCC cell lines and tissues and significantly correlated with the malignancy stage. Furthermore, knockdown of PIWIL2 in a human OSCC cell line Tca8113 induced cell cycle arrest and apoptosis. Silencing PIWIL2 expression also significantly suppressed the migration and invasion abilities of Tca8113 cells.ConclusionsCollectively, our results suggest a functional role of PIWIL2 in regulating OSCC pathogenesis. Our data imply that PIWIL2 could serve as a potential therapeutic target for OSCC treatment.

DLEU1 promotes oral squamous cell carcinoma progression by activating interferon-stimulated genes

Long noncoding RNAs (lncRNAs) are deeply involved in cancer development. We previously reported that DLEU1 (deleted in lymphocytic leukemia 1) is one of the lncRNAs overexpressed in oral squamous cell carcinoma (OSCC) cells, where it exhibits oncogenic activity. In the present study, we further clarified the molecular function of DLEU1 in the pathogenesis of OSCC. Chromatin immunoprecipitation-sequencing (ChIP-seq) analysis revealed that DLEU1 knockdown induced significant changes in the levels of histone H3 lysine 4 trimethylation (H3K4me3) and H3K27 acetylation (H3K27ac) in OSCC cells. Notably, DLEU1 knockdown suppressed levels of H3K4me3/ H3K27ac and expression of a number of interferon-stimulated genes (ISGs), including IFIT1, IFI6 and OAS1, while ectopic DLEU1 expression activated these genes. Western blot analysis and reporter assays suggested that DLEU1 upregulates ISGs through activation of JAK-STAT signaling in OSCC cells. Moreover, IFITM1, one of the ISGs induced by DLUE1, was frequently overexpressed in primary OSCC tumors, and its knockdown inhibited OSCC cell proliferation, migration and invasion. These findings suggest that DLEU1 exerts its oncogenic effects, at least in part, through activation of a series ISGs in OSCC cells.

LncRNA MIR31HG Drives Oncogenicity by Inhibiting the Limb-Bud and Heart Development Gene (LBH) during Oral Carcinoma.

The miR-31 host gene (MIR31HG) encodes a long non-coding RNA (LncRNA) that harbors miR-31 in its intron 2; miR-31 promotes malignant neoplastic progression. Overexpression of MIR31HG and of miR-31 occurs during oral squamous cell carcinoma (OSCC). However, the downstream effectors modulated by MIR31HG during OSCC pathogenesis remain unclear. The present study identifies up-regulation of MIR31HG expression during the potentially premalignant disorder stage of oral carcinogenesis. The potential of MIR31HG to enhance oncogenicity and to activate Wnt and FAK was identified when there was exogenous MIR31HG expression in OSCC cells. Furthermore, OSCC cell subclones with MIR31HG deleted were established using a Crispr/Cas9 strategy. RNA sequencing data obtained from cells expressing MIR31HG, cells with MIR31HG deleted and cells with miR-31 deleted identified 17 candidate genes that seem to be modulated by MIR31HG in OSCC cells. A TCGA database algorithm pinpointed MMP1, BMP2 and Limb-Bud and Heart development (LBH) as effector genes controlled by MIR31HG during OSCC. Exogenous LBH expression decreases tumor cell invasiveness, while knockdown of LBH reverses the oncogenic suppression present in MIR31HG deletion subclones. The study provides novel insights demonstrating the contribution of the MIR31HG-LBH cascade to oral carcinogenesis.

A study of epithelial-mesenchymal transition immunohistochemical markers in primary oral squamous cell carcinoma

To analyze markers of epithelial-mesenchymal transition (EMT) in oral squamous cell carcinoma (SCC) using E-cadherin, epithelial membrane antigen (EMA), vimentin, and smooth muscle actin (SMA), and their association with tumor metastasis and grade. This was a retrospective study, which included 45 diagnosed cases of primary oral SCC with known lymph node status, taken from the archives of the Department of Pathology along with their clinical profile. Histomorphologic evaluation and immunohistochemical (IHC) analysis for E-cadherin, EMA, vimentin, and SMA were performed. IHC expression of these markers was compared with tumor differentiation as well as lymph node metastasis. We observed that reduced E-cadherin expression and positive expression of SMA were significantly higher in tumors having lymph node metastasis and loss of tumor differentiation, respectively. Reduced EMA expression was associated with the presence of lymph node metastasis but not with the histologic differentiation. Vimentin positivity did not show any correlation with lymph node metastasis or histologic differentiation. This study emphasizes the importance of EMT in the pathogenesis of oral SCC. The use of biomarkers like E-cadherin, EMA, and SMA might be a valuable tool for predicting patient outcomes and therapy.

Infrared Microspectroscopy With Multivariate Analysis to Differentiate Oral Hyperplasia From Squamous Cell Carcinoma: A Proof of Concept for Early Diagnosis.

Despite having numerous advances in therapeutics, mortality and morbidity due to oral cancer incidence are still very high. Early detection can improve the chances of survival in most patients. However, diagnosis at early stages can be challenging as premalignant conditions are usually asymptomatic. Currently, histological assessment remains the gold standard for diagnosis. Early diagnosis poses challenges to pathologists due to less severe morphological changes associated with early stages. Therefore, a fast and robust method of detection based on molecular changes is needed for early diagnosis. © 2021 Wiley Periodicals LLC. In the present study, Fourier transform infrared (FTIR) spectroscopic imaging has been used to differentiate early-stage oral hyperplasia from adjacent normal (AN) and oral squamous cell carcinoma (OSCC). Hyperplasia is often considered as an initial event in the pathogenesis of oral cancer and OSCC is the most common advanced stage of malignancy. Differentiating normal versus hyperplasia and hyperplasia versus OSCC can remain quite challenging on occasion using conventional staining as the histological assessment is based on morphological changes. Unsupervised hierarchical cluster analysis (UHCA) has been performed on FTIR images of multiple tissues together that provided some degree of classification among tissue groups. The AN epithelium clustered distinctively using UHCA from both hyperplasia and grades 1 and 2 of OSCC. An increase in the content of DNA, denaturation of protein, and altered lipid structures were more clearly elucidated with spectral analysis. This study demonstrates a simple strategy to differentiate early-stage oral hyperplasia from ANand OSCC using UHCA. This study also proposes a future alternative method where FTIR imaging can be used as a diagnostic tool for cancer at early stages.

MicroRNA‑198 suppresses tumour growth and metastasis in oral squamous cell carcinoma by targeting CDK4.

MicroRNAs (miRNAs/miR) often contribute to the progression of oral squamous cell carcinoma (OSCC) via the regulation of mRNA. The present study aimed to investigate the role of miR-198 in OSCC pathogenesis and explore the underlying mechanism. Reverse transcription-quantitative (RT-q)PCR was performed to determine miR-198 expression in OSCC tissues and cell lines, and univariate and multivariate analyses were applied to evaluate the survival of patients with OSCC. The effects of miR-198 on OSCC cell lines were studied in vitro and in vivo. A set of epithelial-mesenchymal transition (EMT) markers were detected to determine whether miR-198 was involved in EMT. Lastly, using luciferase assays, a novel target of miR-198 was identified and the effect of the new target gene of miR-198 on cell proliferation and invasion was also studied. It was identified that miR-198 expression was decreased in OSCC tissues and cell lines, and low expression of miR-198 was associated with poor overall survival and disease-free survival. Overexpression of miR-198 appeared to significantly inhibit the proliferation, invasion and EMT of OSCC cells. Moreover, the luciferase assay results showed that miR-198 interacted with cyclin-dependent kinase 4 (CDK4) by directly targeting the miRNA-binding site in the CDK4 sequence, and RT-qPCR results showed that CDK4 expression was increased in OSCC tissues and cell lines. In addition, transfection of small interfering RNA against CDK4 in OSCC cells showed similar inhibitory effects on cell proliferation, invasion and EMT, whereas CDK4 overexpression in OSCC cells partially reversed the inhibitory effects of the miR-198 mimic. The present results indicated that miR-198 suppressed OSCC tumour growth and metastasis by directly targeting CDK4 expression. Thus, miR-198 may be a potential therapeutic target in the treatment of OSCC.

Salivary Porphyromonas gingivalis predicts outcome in oral squamous cell carcinomas: a cohort study

BackgroundStudies suggest Porphyromonas gingivalis (Pg) increased the incidence of oral squamous cell carcinoma (OSCC). However, fimA genotypes distribution of Pg, the origination of Pg in tissue, and its prognostic value are inconclusive. We aimed to investigate the frequency of fimA genotypes in OSCC patients, study the association between Pg and OSCC, and explore the prognostic value of Pg.MethodsThe abundance of Pg in saliva from the OSCC group and the OSCC-free group was analysed by qPCR. The presence of Pg was explored in OSCC tissue and para-cancerous tissue by in situ hybridization. The frequency of fimA genotypes in saliva and OSCC tissue was determined by PCR, then PCR products were sequenced and compared. Clinical data were extracted, and patients followed up for a median period of 23 months. Clinicopathological variables were compared with the abundance of Pg using Pearson Chi-square test or Fisher’s exact test. The disease-free survival (DFS) rate was calculated by Kaplan–Meier method with log-rank tests.ResultsComparing the OSCC-free group, 95 patients with OSCC showed a high abundance of Pg in saliva (P = 0.033), and OSCC tissue showed strong in situ expression of Pg compared with paired normal tissue. Patients with OSCC showed a dominant distribution of Pg with genotype I + Ib (21.1%), II (31.6%), and IV (21.1%). FimA genotypes detected in saliva were in accordance with those in OSCC tissue, there was, moreover, a significant similarity in amplified Pg fragments. Of the 94 responsive OSCC patients, the recurrence rate was 26.6% (25/94). Overabundance of Pg in saliva showed advanced pathologic staging (P = 0.008), longer disease-free time (P = 0.029) and lower recurrence rate (P = 0.033). The overabundance of Pg in saliva was associated with improved disease-free survival (P = 0.049).ConclusionsThis study indicated that Pg might involve in the pathogenesis of OSCC, Pg carrying fimA I, Ib, II, and IV were prevalent genotypes in patients with OSCC, the provenance of Pg in OSCC tissue might be from the salivary microbial reservoir, and the abundance of Pg in saliva might consider as a favorable potential prognostic indicator in OSCC.

Associations of high-resolution-typing-defined MICA and MICB polymorphisms, and the levels of soluble MICA and MICB with Oral Squamous Cell Carcinoma in Bulgarian patients.

Oral Squamous Cell Carcinoma (OSCC) is a malignancy characterized by an aggressive tumor growth and significant mortality. Clarifying mechanisms responsible for immunomodulation are among the main challenges for the development of personalized approaches for the management of patients with Oral Squamous Cell Carcinoma. The aim of the present study was to analyze the relevance of MICA and MICB to Oral Squamous Cell Carcinoma pathogenesis focusing on allele polymorphisms and the levels of soluble MICA and MICB molecules. 73 patients diagnosed with Oral Squamous Cell Carcinoma and 149 healthy controls from the Bulgarian population were included in the study. MICA and MICB polymorphism was analyzed at high-resolution level using Next-Generation Sequencing. Serum levels of soluble MICA and MICB molecules were measured by ELISA. Our results show significant protective association with MICB*002:01, while relatively rare alleles MICB*018, *019, and *020 were observed with statistically significant increased frequency in Oral Squamous Cell Carcinoma patients compared to controls. Additionally, a predisposing association was observed for MICA*008:01-MICB*019 haplotype. A correlation analysis between functionally relevant MICA polymorphisms and sMICA showed that homozygosity for MICA-A5.1 or 129Val in OSCC patients was associated with significantly higher serum levels of sMICA. This is the first study showing significant associations between MICB alleles and Oral Squamous Cell Carcinoma and suggesting the possible role of MICB in immunosurveillance in Oral Squamous Cell Carcinoma development. Observed correlations between the levels of soluble MICA molecules and functionally relevant polymorphisms might represent a further step toward a better understanding of molecular mechanisms of Oral Squamous Cell Carcinoma and developing strategies for therapeutic targeting harnessing effective immunosurveillance.

Integrated analysis and identification of nine-gene signature associated to oral squamous cell carcinoma pathogenesis.

The online version contains supplementary material available at 10.1007/s13205-021-02737-4.

NCAPG, mediated by miR-378a-3p, regulates cell proliferation, cell cycle progression, and apoptosis of oral squamous cell carcinoma through the GSK-3β/β-catenin signaling.

Exploring the molecular mechanism of oral squamous cell carcinoma (OSCC) pathogenesis is of great significance for its improvement and therapy. Non-structural maintenance of chromatin condensin I complex subunit G (NCAPG) is responsible for chromatin condensation and is associated with the progression of many malignant tumors. This study was aimed to investigate the role of NCAPG on OSCC pathogenesis. NCAPG mRNA expression data in OSCC tissues were obtained from the Gene Expression Omnibus (GEO) database and NCAPG protein expression in OSCC cell lines was determined by western blotting analysis. The results demonstrated that NCAPG expression in OSCC tissues and cells was higher than that of normal control. Following the short interfering RNA (siRNA) knockdown of NCAPG in two OSCC cell lines, we observed that NCAPG depletion notably inhibited OSCC proliferation and cell cycle progression, as well as promoted apoptosis in vitro. Besides, silencing of NCAPG specifically inhibited the GSK-3β/β-catenin signaling. Furthermore, we demonstrated that NCAPG was a downstream target of miR-378a-3p. NCAPG silencing counteracted the effect of the miR-378a-3p inhibitor on cell proliferation/cycle induction. Collectively, these findings suggest that NCAPG is crucial in OSCC progression and development, and may serve as a potential therapeutic target for OSCC.

Expression Analysis of ALDH1A1 and ALDH1A3 Genes in Oral Squamous Cell Carcinoma Patients

Oral squamous cell carcinoma (OSCC) is still one of the leading cancer related death, worldwide. Owing to the critical role of ALDH1 isoforms in pathogenesis of cancers and controversies in previous reports, the present study was conducted to determine the expression of two challenging ALDH1 isoforms in OSCC tissue samples. RNA was isolated from 30 OSCC and normal fresh tissues margins and then was converted to cDNA using Thermo Scientific™ Fermentas cDNA synthesis kit. Reverse Transcription Real time polymerase chain reaction (RT-Real time PCR) was performed on synthesized cDNA using primer pairs specific to ALDH1A1 and ALDH1A3 genes. It was found that the ALDH1A1 mRNA expression was significantly higher in OSCC samples compared to normal margins (P-value= 0.001). ALDH1A1 and ALDH1A3 expression was significantly associated with alcohol drinking (P-value= 0.01) and smoking (P-value=0.008), respectively. There was no correlation among ALDH1A1 and ALDH1A3 expressions and clinicopathological features (p-value>0.05). Confirmation of the over-expression of ALDH1A1 isoform in OSCC samples maybe indicating that it plays critical role in pathogenesis of OSCC. However, owing to the discrepant findings on the association of ALDH1A1 expression with clinicopathological features, further studies are required to identify its role as driver or passenger carcinogenic alteration and its exact association with smoking.

A systematic review of specific pathways in oral squamous cell carcinoma

Background Oral squamous cell carcinoma (OSCC) eludes all therapeutic modalities because its etiological factors, molecular mechanisms, and pathways of carcinogenesis remain controversial. Objective This systematic review aims to identify and investigate the specific pathways commonly documented in the published research on OSCC. Results The most frequently studied pathways in OSCC, according to the reviewed molecular studies, are p53 tumor suppressor, collective Serine/Threonine Kinases, transcription factor NF-kB, integrins and cell proliferation, vascular endothelial growth factor, Wnt signaling, the PI3K/AKT pathway, cadherins and adhesion of epithelial cells and cyclooxygenase 2 pathways. However, the MEK/ERK pathway is computationally detected as a less frequent oncogenic pathway in OSCC. Advances in Knowledge Although the former pathways have been extensively studied, other pathways are involved in the pathogenesis of OSCC, such as Notch signaling, cell death via Fas or TNFR1 Receptors, and the p38 MAPK pathway, remain understudied in OSCC. Future studies elucidating the contribution of these pathways in the development of OSCC are urgently required.

Expression Profile of Circular RNAs in Oral Squamous Cell Carcinoma.

BackgroundCircular RNAs (circRNAs) are involved in the pathogenesis of several diseases. Among oral maxillofacial cancers, oral squamous cell carcinoma (OSCC) has the highest incidence. However, the role of circRNAs in OSCC is still not clear. The aim of our study was to evaluate the circRNA expression profile in OSCC and explore further the potential role of circRNAs in the pathogenesis of OSCC.MethodsCircRNA sequencing was performed in 6 pairs of samples of OSCC and normal oral mucosal tissues. Expression of selected circRNAs was validated by qRT-PCR. GO and KEGG analyses were performed and binding relationships between circRNAs and miRNAs were predicted. The hsa_circ_0001766/miR-877-3p/VEGFA axis was selected to further elucidate its role in OSCC.ResultsWe showed that there were 122 differentially expressed (DE) circRNAs. Eight out of 10 selected circRNAs were validated by qRT-PCR. GO and KEGG analyses indicated that the identified DE circRNAs might be involved in the progression of OSCC. Then, after identification by Sanger sequencing and RNase R assay, the upregulated hsa_circ_0001766 was selected to investigate its potential role in OSCC. Bioinformatics analysis showed that hsa_circ_0001766 might act as a competing endogenous RNA (ceRNA) that sponged miR-877-3p to upregulate VEGFA expression. We selected OSCC cell lines SCC9 and SCC25. PCR results showed that the expression of hsa_circ_0001766 and VEGFA was upregulated in SCC9 and SCC25. Subsequently, using western blot, PCR, CCK8, and colony formation assays, we found that knocking down circRNA0001766 inhibited the expression of VEGFA and the proliferation of OSCC cells. Following this, miR-877-3p inhibitor reversed the inhibitory effect of si-hsa_circ_0001766 on expression of VEGFA and proliferation of OSCC cells.ConclusionsIn conclusion, our study revealed the possible role of circRNAs in the pathogenesis of OSCC, and identified the potential role of the hsa_circ_0001766/miR-877-3p/VEGFA axis in OSCC progression.

RGS12 Represses Oral Cancer via the Phosphorylation and SUMOylation of PTEN

Oral squamous cell carcinoma (OSCC) is the most common head and neck cancer characterized by aggressive local invasion and metastasis. The pathogenesis of OSCC is mainly due to the accumulation of genetic alterations in epithelial cells, but the underlying mechanism for its development remains unclear. Here, we found that the expression level of regulator of G protein signaling 12 (RGS12) was significantly reduced in human OSCC. To understand the role and mechanism of RGS12 in OSCC, we generated a novel RGS12 global knockout (CMVCre/+; RGS12fl/fl) mouse model by crossing RGS12fl/fl mice with CMV-Cre transgenic mice and then further induced the mice to develop OSCC by using 4-nitroquinoline 1-oxide (4NQO). Deletion of RGS12 exhibited aggressive OSCC in the tongue compared with the control RGS12fl/fl mice. Knockdown of RGS12 in OSCC cells significantly increased cell proliferation and migration. Mechanistically, we found that RGS12 associated with phosphatase and tension homolog (PTEN) via the PDZ domain to upregulate the phosphorylation and SUMOylation of PTEN and then correspondingly inactivated the AKT/mTOR signaling pathway. To test the potential therapeutic effect of RGS12 on OSCC, we overexpressed RGS12 in OSCC cells and found a significant inhibition of cancer cell proliferation and migration. Moreover, subcutaneous inoculation of RGS12-overexpressed OSCC cells in NOD scid mice showed a significant reduction in tumor formation. Our findings reveal that RGS12 is an essential tumor suppressor and highlights RGS12 as a potential therapeutic target and prognostic biomarker of OSCC.

Inhibition of CAL27 Oral Squamous Carcinoma Cell by Targeting Hedgehog Pathway With Vismodegib or Itraconazole.

Oral Squamous Cell Carcinoma (OSCC) presents an important challenge for the health systems worldwide. Thus, unraveling the biological mechanisms involved in OSCC pathogenesis is essential to the discovery of new drugs with anticancer potential. The Hedgehog (HH) pathway has shown promising results as a therapeutic target both in vitro and in vivo. This study aimed to investigate the effects of vismodegib and itraconazole on the expression of Hedgehog (HH) genes (PTCH1, SMO, and GLI1), cell cycle and cell death in OSCC cells. Alamar Blue assay was used to assess the cytotoxicity of vismodegib and itraconazole in a panel of oral cancer cell lines, including CAL27. The expression of HH signaling components after treatment with vismodegib and itraconazole, at concentrations of 25 or 50 μg/ml was evaluated by qPCR. Cell cycle and apoptosis were evaluated by flow cytometry after 72 h treatment with 50 μg/ml of vismodegib or itraconazole. HH signaling was activated in OSCC cell lines CAL27, SCC4, SCC9, and HSC3. Vismodegib and itraconazole significantly reduced CAL27 cell viability after 48 h of treatment. Gene expression of PTCH1, SMO, and GLI1 decreased in response to 24 h of treatment with vismodegib or itraconazole. Furthermore, CAL27 cells exhibited alterations in morphology, cell size, and cellular granularity. An increase in the DNA fragmentation was observed after treatment and both inhibitors induced apoptosis after 72 h. In conclusion, SMO inhibitors vismodegib and itraconazole demonstrably reduced the expression of HH genes in CAL27 OSCC cell line. In addition, treatment with vismodegib and itraconazole reduced cellular viability and altered the morphology of CAL27 cells, and also induced apoptosis.

Significance of P16INK4A Expression and PTEN Loss of Heterozygosity in Human Papilloma Virus-related Oral Squamous Cell Carcinoma.

p16 and PTEN are tumor suppressor genes. Loss of these molecules in oral squamous cell carcinoma (OSCC) has been studied worldwide. In this study, we explored whether p16 cooperates with inactive PTEN during the pathogenesis of OSCC, especially in regard to tumor aggressiveness and proliferation. Immunocytochemistry and western blot analysis were used to examine the levels of p16 and PTEN. Sequencing analysis was performed to identify mutations in the PTEN gene and HPV infection. Fluorescence in situ hybridization was used to examine the presence of the PTEN locus. PTEN analysis showed high positivity in T4 samples. HPV-positive tumors correlated with tabagism, tumor size 3 and 4, disease stages 3 and 4, presence of lymph node metastasis (N1) and poor differentiation. Immunoexpression of p16 was strongly correlated with the presence of HPV. PTEN demonstrated a higher reactivity in advanced disease stages and p16 was strongly associated with HPV. Viral presence decreases tumor aggressiveness. Patients with advanced stage lesions demonstrated lower survival rate.

Current Understanding of the HIF-1-Dependent Metabolism in Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is the 10th most frequent human malignancy and is thus a global burden. Despite some progress in diagnosis and therapy, patients’ overall survival rate, between 40 and 55%, has stagnated over the last four decades. Since the tumor node metastasis (TNM) system is not precise enough to predict the disease outcome, additive factors for diagnosis, prognosis, prediction and therapy resistance are urgently needed for OSCC. One promising candidate is the hypoxia inducible factor-1 (HIF-1), which functions as an early regulator of tumor aggressiveness and is a key promoter of energy adaptation. Other parameters comprise the composition of the tumor microenvironment, which determines the availability of nutrients and oxygen. In our opinion, these general processes are linked in the pathogenesis of OSCC. Based on this assumption, the review will summarize the major features of the HIF system-induced activities, its target proteins and related pathways of nutrient utilization and metabolism that are essential for the initiation, progression and therapeutic stratification of OSCC.