Tongue Squamous Cell Carcinoma Cells Research Articles

Regulating Lipid Metabolism via Mitochondrial Dynamics in Tongue Squamous Cell Carcinoma Cancer Stem Cells.

Cancer stem cells (CSCs) are a sub-population of cancer cells present in many kinds of malignant tumors that have the potential for self-proliferation and differentiation. These cells have been demonstrated as the main cause of tumor recurrence and metastasis. Strong evidence indicates that CSCs prefer reprogrammed fatty acid β-oxidation over oxidative phosphorylation for sustaining energy supply. Although mitochondrial dynamics participate in the regulation of cancer stemness, the correlation between the inhibition of mitochondrial fission and the regulation of lipid metabolism in CSCs remains poorly understood. The human tongue squamous cell carcinoma (TSCC) cell lines CAL27 and SAS were used to obtain the CSCs by 3D Spheroid Culture. Then,western blot methods, RT-PCR and flow cytometry analysis were used to identify the TSCC CSCs. Next, Immunofluorescence method, transmission electron microscopy detection and western blot methods were used to evaluate the mitochondrial morphology and the quantity of lipid droplets (LDs). Lastly, lipidomic analysis was applied to explored the lipidomic alterations of TSCC CSCs with different mitochondrial morphology. Here, we show that the quantity of lipid droplets containing intracellular triglyceride (TG) can be decreased by regulating mitochondrial morphology. Lipidomic analysis using ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) also compared alterations in lipid metabolites in tongue squamous cell carcinoma (TSCC) CSCs, TSCC cells (non-CSCs), and CSCs with different mitochondrial morphology. Discriminant lipids of statistical significance were successfully annotated, including phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), sphingomyelins (SMs), triacylglycerols (TGs), phosphatidylglycerols (PGs), phosphatidylserines (PSs), lysophosphatidylcholines (LPCs), and lysophosphatidylethanolamines (LPEs). This study provides a deeper insight into the alterations of lipid metabolism associated with TSCC CSCs, non-CSCs and CSCs regulated by mitochondrial dynamics and thus serves as a guide toward novel targeted therapies.

Capsaicin exerts synergistic pro-apoptotic effects with cisplatin in TSCC through the calpain pathway via TRPV1.

Capsaicin (CAP) exerts significant anti-tumor effects on a variety of tumors, with low intrinsic toxicity. Cisplatin (DDP) is currently the first-line drug for the treatment of oral cancer; however, its clinical efficacy is impeded by chemoresistance and negligible side effects. Whether the combined use of CAP and DDP has a synergistic antitumor effect on tongue squamous cell carcinoma (TSCC) cells and its underlying mechanisms remains unclear. The present study revealed that CAP reduced the activity of TSCC cells in a dose- and time-dependent manner. We also observed changes in the mitochondrial functional structure of TSCC cells, along with the induction of mitochondrial apoptosis. Moreover, when CAP was combined with DDP, a synergistic cytotoxic effect on TSCC cells was observed, which had a significant impact on inducing apoptosis, inhibiting proliferation, and disrupting the mitochondrial membrane potential in TSCC cells compared to the single-drug treatment and control groups. These effects are associated with TRPV1, a high-affinity CAP receptor. The combined use of CAP and DDP can activate the TRPV1 receptor, resulting in intracellular Ca2+ overload and activation of the calpain pathway, ultimately leading to mitochondrial apoptosis. This potential mechanism was validated in TSCC xenograft models. In conclusion, our findings clearly demonstrate that CAP exerts synergistic pro-apoptotic effects with DDP in TSCC through the calpain pathway mediated by TRPV1. Thus, CAP can be considered an effective adjuvant drug for DDP in the treatment of TSCC.

Actin filament-associated protein 1-antisense RNA1 promotes the development and invasion of tongue squamous cell carcinoma via the AFAP1-AS1/miR-133a-5p/ZIC2 axis.

The present study aimed to explore the biological role and underlying mechanism of the long non-coding RNA actin filament-associated protein 1-antisense RNA1 (lncRNA AFAP1-AS1) in the progression of tongue squamous cell carcinoma (TSCC). A quantitative reverse transcriptase-PCR (RT-qPCR) was conducted to assess relative levels of the miR-133a-5p, lncRNAs AFAP1-AS1 and zinc finger family member 2 (ZIC2) in TSCC cell lines and specimens, whereas ZIC2 protein levels were measured using western blotting. After modifying the levels of expression of lncRNA AFP1-AS1, miR-133a-5p and ZIC2 using lentivirus or plasmid transfection, we examined AKT/epithelial-mesenchymal transition signaling pathway alterations, in vivo carcinogenesis of TSCC in nude mice and in vitro malignant phenotypes. A dual-luciferase reporter assay was conducted to confirm the targeting relationship between ZIC2 and miR-133a-5p, as well as between miR-133a-5p and lncRNA AFAP1-AS1. Based on The Cancer Genome Atlas (TCGA) database, we additionally validated AFP1-AS1. The potential biological pathway for AFP1-AS1 was investigated using gene set enrichment analysis (GSEA). We also evaluated the clinical diagnostic capacities of AFP1-AS1 and clustered the most potential biomarkers with the Mfuzz expression pattern. Finally, we also made relevant drug predictions for AFP1-AS1. In TSCC cell lines and specimens, lncRNA AFAP1-AS1 was upregulated. ZIC2 was upregulated in TSCC cells as a result of lncRNA AFAP1-AS1 overexpression, which also promoted TSCC cell migration, invasion, viability, and proliferation. Via the microRNA sponge effect, it was found that lncRNA AFAP1-AS1 could upregulate ZIC2 by competitively inhibiting miR-133a-5p. Interestingly, knockdown of ZIC2 reversed the biological roles of lncRNA AFAP1-AS1 with respect to inducing malignant phenotypes in TSCC cells. In addition, in vivo overexpression of lncRNA AFAP1-AS1 triggered subcutaneous tumor growth in nude mice implanted with TSCC cells and upregulated ZIC2 in the tumors. The TCGA database findings revealed that AFAP1-AS1 was significantly upregulated in TSCC specimens and had good clinical diagnostic value. The results of GSEA showed that peroxisome proliferator-activated receptor signaling pathway was significantly correlated with low expression of AFP1-AS1. Finally, the results of drug prediction indicated that the group with high AFAP1-AS1 expression was more sensitive to docetaxel, AZD4547, AZD7762 and nilotinib. The upregulation of lncRNA AFAP1-AS1, which increases TSCC cell viability, migration, proliferation and invasion via the AFAP1-AS1/miR-133a-5p/ZIC2 axis, aids in the progression of TSCC.

In Vitro and In Vivo Analyses Reveal Tumor-Derived Exosome miR-558 Promotes Angiogenesis in Tongue Squamous Cell Carcinoma by Targeting Heparinase.

This study aimed to investigate the role of miR-558 in tumor angiogenesis by targeting heparinase (HPSE) in tongue squamous cell carcinoma (TSCC)-derived exosomes. In the present study, the role of exosome miR-558 in angiogenesis in vitro and in vivo was investigated by cell proliferation, migration, tube formation, subcutaneous tumor formation in mice, and in vivo Matrigel plug assay. The target genes of miR-558 were detected by means of dual luciferase assay. It was found that TSCC cells secrete miR-558 into the extracellular environment, with exosome as the carrier. Human umbilical vein endothelial cells (HUVEC) ingested exosomes, which not only increased the expression level of miR-558, but also enhanced their proliferation, migration, and tube formation functions. In vivo Matrigel plug assay demonstrated that TSCC cell-derived exosome miR-558 promoted neovascularization in vivo. Compared with negative control cells, TSCC cells overexpressing miR-558 formed subcutaneous tumors in nude mice, with larger volume, heavier mass, and more vascularization. Dual luciferase assay confirmed that HPSE was the direct target gene regulated by miR-558. HPSE promoted the proliferation, migration, and tube formation of HUVECs, and the knockout of HPSE could downregulate the pro-angiogenic effect of miR-558. In summary, miR-558 in TSCC exosomes promotes the proliferation, migration, and tube formation of HUVECs by targeting HPSE, and enhancing tumor angiogenesis.

Knockdown of LRP5 Promotes Proliferation and Invasion of Tongue Squamous Cell Carcinoma through Compensatory Activation of Akt Signaling.

The role of LRP5, a critical receptor in the Wnt signaling pathway, remains unexplored in tongue squamous cell carcinoma (TSCC). This study investigates the impact of LRP5 knockdown on the biological behaviors of TSCC cell lines both in vitro and in vivo. Our findings indicate that LRP5 knockdown significantly enhances cell proliferation, migration, and invasion in CAL27 and SCC25 cell lines. RNA-seq analysis reveals compensatory activation of the Akt pathway, with 119 genes significantly upregulated post-LRP5 knockdown. Elevated MMP1 expression suggests its potential involvement in TSCC progression. Western blot analysis demonstrates increased Akt phosphorylation, upregulated proliferation-related PCNA, and downregulated apoptosis-related caspase-3 after LRP5 knockdown. Down-regulation of E-cadherin and β-Catenin, proteins associated with cell adhesion and invasion, further elucidates the molecular mechanism underlying increased cell migration and invasion. Our study concludes that compensatory Akt pathway activation is essential for the LRP5 knockdown-induced migration and proliferation of CAL27 and SCC25 cells. These results highlight LRP5 as a potential therapeutic target for TSCC. Simultaneous inhibition of Wnt and Akt signaling emerges as a promising approach for TSCC treatment.

Investigating whether exosomal miR-205-5p derived from tongue squamous cell carcinoma cells stimulates the angiogenic activity of HUVECs by targeting AMOT.

Although exosomal microRNAs (exo-miRNAs) regulate angiogenesis, they are not sufficient for the development of anti-vascular drugs for tongue squamous cell carcinoma (TSCC). miR-205-5p is an exo-miRNA that is highly expressed in the saliva of patients with oral SCC. We aimed to clarify the role and molecular mechanism of exosomal miR-205-5p in regulating TSCC angiogenesis. Effect of exosomes derived from TSCC cells on human umbilical vein endothelial cell (HUVEC) function was determined using the CCK-8, Transwell, Transwell-Matrigel, and Matrigel-based tube formation assays. Protein levels were detected by western blot. The binding between miR-205-5p and the 3'UTR of AMOT was verified using a luciferase reporter assay. Exosomal miR-205-5p (exo-miR-205-5p) promoted the proliferation, migration, and invasion of HUVECs, increased the number of tubes formed by HUVECs, and increased the vascular endothelial growth factor receptor 2 levels in HUVECs. Exo-miR-205-5p downregulated the AMOT level in HUVECs. Results of the luciferase reporter assay showed that miR-205-5p could bind to the 3'UTR of AMOT. AMOT overexpression blocked the effect of exo-miR-205-5p on HUVEC functions. Exo-miR-205-5p derived from TSCC regulates the angiogenic activity of HUVECs by targeting AMOT and might be a new molecular target for the development of anti-vascular drugs for TSCC.

Silencing of heat shock factor 1 (HSF1) inhibits proliferation, invasion, and epithelial-mesenchymal transition in oral squamous cell carcinoma.

Oral squamous cell carcinoma is characterized by high rates of morbidity and mortality. Evidence obtained for different types of cancer shows that tumor initiation, progression, and therapeutic resistance are regulated by heat shock factor 1. This research aimed to analyze the effects of heat shock factor 1 on the biological behavior of oral squamous cell carcinoma. Clinicopathological and immunoexpression study of heat shock factor 1 in 70 cases of oral tongue SCC and functional assays by gene silencing of this factor in an oral tongue SCC cell line. Heat shock factor 1 was overexpressed in oral tongue SCC specimens compared to normal oral mucosa (p < 0.0001) and in the SCC15 line compared to immortalized keratinocytes (p < 0.005). No significant associations were observed between overexpression of heat shock factor 1 and clinicopathological parameters or survival rates of the oral tongue SCC cases in the present sample. In vitro experiments showed that heat shock factor 1 silencing inhibited cell proliferation (p < 0.005) and cell cycle progression, with the accumulation of cells in the G0/G1 phase (p < 0.01). In addition, heat shock factor 1 silencing reduced cell invasion capacity (p < 0.05) and epithelial-mesenchymal transition, characterized by a decrease in vimentin expression (p < 0.05) and an increase in E-cadherin expression (p < 0.001). Heat shock factor 1 may exert several functions that help maintain cell stability under the stressful conditions of the tumor microenvironment. Thus, strategies targeting the regulation of this protein may in the future be a useful therapeutic tool to control the progression of oral squamous cell carcinoma.

Brucea javanica oil inhibits tongue squamous cell invasion and metastasis by regulating miR-138-EZH2 pathway

Tongue squamous cell carcinoma (TSCC) is one of the most common malignant tumors of head and neck. Its incidence is on the rise, and the proportion of young patients is gradually increasing, which is prone to tumor recurrence and metastasis. At present, there is no effective method to completely treat TSCC. Studies have shown that brucea javanica oil (BJO) has good antitumor activity against lung cancer and gastrointestinal tumors, but its therapeutic effect on TSCC is not clear. We have previously confirmed that oleic acid, the main component of BJO, can induce apoptosis of TSCC and reduce its invasion and metastasis ability. However, the anticancer effect and mechanism of BJO in TSCC remain unclear. In order to further explore the effects of BJO on the biological characteristics of TSCC cells, we studied the effects of different concentrations of BJO on the migration, invasion ability and epithelial mesenchymal transition (EMT) progression of TSCC cells and the possible mechanisms through in vitro experiments. We found that BJO could inhibit the invasion and metastasis of TSCC and up-regulate miR-138. After BJO treatment, the expression of E-cad was significantly increased, while the expression of EZH2, Slug, p-ERK1/2 and Vimentin was significantly decreased. EZH2 is a miR-138 target gene involved in TSCC. BJO inhibits TSCC invasion and metastasis by regulating the miR-138-EZH2 pathway. In vivo experiments have also well demonstrated the targeting effect of this pathway. This study provides a new therapeutic strategy for the treatment of TSCC.

ARNTL inhibits the malignant behaviors of oral cancer by regulating autophagy in an AKT/mTOR pathway-dependent manner.

Current studies have shown that ARNTL, an important clock gene, plays an anticancer role and is downregulated in certain types of cancer. However, the biological functions and mechanisms of ARNTL in tumors remain largely unknown. This study aimed to elucidate how ARNTL-induced autophagy impacts the biological properties of tongue squamous cell carcinoma (TSCC) cells and the mechanisms by which ARNTL expression activates autophagy. ARNTL was stably overexpressed in TSCC cells to investigate its functions in vitro and in vivo. We found that activation of autophagy induced by ARNTL decreases cell proliferation, enhances cell death, and hinders the migratory ability of TSCC cells. Moreover, ARNTL antagonizes the AKT/mTOR pathway, which potentiates autophagy induction. The manipulation of Akt activation cancels the effects of ARNTL overexpression on the biological behaviors of TSCC cells. Furthermore, after the addition of SC79, the upregulated BAX expression due to ARNTL overexpression and downregulated expressions of BCL-2 and MMP2 were remarkably rescued. In vivo tumorigenicity assays and immunohistochemistry also confirmed that ARNTL overexpression suppresses tumor development. Our study found for the first time that ARNTL inhibits the malignant behaviors of oral cancer cells by regulating autophagy in an AKT/mTOR pathway-dependent manner, which provides a novel theoretical basis for the potential future application of ARNTL to treat patients with oral cancer.

KLK4 affects the biological behavior of oral squamous cell carcinoma through Wnt/β catenin and PI3K/Akt signaling pathways

Purpose: To investigate the effect of kallikrein 4 (KLK4) on oral squamous cell carcinoma (OSCC), and its mechanism of action.&#x0D; Method: Human tongue SCC cell line TCA-8113 was cultured, and the interference fragment and irrelevant sequence fragment of KLK4 gene were designed and synthesized. These fragments were constructed into pgcsi-h1 plasmid. Then, the TCA-8113 cells were cultured for 24 h, followed by transfection, after which TCA-8113-NC and TCA-8113-siKLK4 cells were obtained. The mRNA expression of KLK4 was determined using quantitative real-time-polymerase chain reaction (PCR). Proliferative potential was assessed using MTT method, while apoptosis was determined by flow cytometry. Furthermore, cell migration was evaluated by scratch test, and Transwell cell invasion experiment was used to determine cell invasion. Immunoblot assay was applied to determine the expressions of E- and N-cadherins, vimentin, uPA, Wnt1, β-Catenin, p-pi3k and p-Akt.&#x0D; Result: Compared with TCA-8113-nc, the expression of KLK4 in TCA-8113-siKLK4 cells was significantly down-regulated, while, relative to TCA-8113-NC, TCA-8113-siKLK4 cells had significantly reduced cell proliferation, invasion and migration, and enhanced apoptosis (p &lt; 0.05). Moreover, there was significant up-regulation of E- and N-cadherins, vimentin, uPA, Wnt1, β-Catenin, p-pi3k and p-Akt in TCA-8113-siKLK4 group, relative to TCA-8113-NC (p &lt; 0.05).&#x0D; Conclusion: KLK4 enhances the multiplication, metastasis and invasive potential of OSCC cells, but suppresses apoptotic changes via activation of Wnt/β-catenin/PI3K/Akt signal route. The potential of Kallikrein-4 as biomarker for OSCC cells should be determined in suitable animal models.

Expression and effect of heterogeneous nuclear ribonucleoprotein A2/B1 in tongue squamous cell carcinoma.

Tongue squamous cell carcinoma (TSCC) is a common cancer in the oral and maxillofacial region, which seriously endangers people's life and health.Heterogeneous nuclear ribonucleoprotein A2/B1(hnRNP A2/B1) is an RNA-binding protein that regulates the expression of a variety of genes and participates in the occurrence and development of a variety of cancers. This study aims to investigate the role of hnRNP A2/B1 in TSCC progression. The differential expression of hnRNP A2/B1 in oral squamous cell carcinoma (OSCC) and normal oral mucosa cells and tissues was analyzed based on the gene expression profiles of GSE146483 and GSE85195 in the Gene Expression Omnibus (GEO) database. The correlation between hnRNP A2/B1 expression and disease-free survival of TSCC patients was analyzed based on TSCC related chip of GSE4676. TSCC cancer and paracancerous tissue samples of 30 patients were collected in Hunan Cancer Hospital from July to December 2021. Real-time RT-PCR and Western blotting were used to verify the mRNA and protein expression of hnRNP A2/B1 in TSCC patients'samples, respectively. Human TSCC Tca-8113 cells were transfected with hnRNP A2/B1 empty vector (a sh-NC group), knockdown plasmid (a sh-hnRNP A2/B1 group), empty vector overexpression plasmid (an OE-NC group) and overexpression plasmid (an OE-hnRNP A2/B1 group), respectively. The knockdown or overexpression efficiency of hnRNP A2/B1 was detected by Western blotting. The proliferation activity of Tca-8113 cells was detected by cell counting kit-8 (CCK-8), and the apoptosis rate of Tca-8113 cells was detected by flow cytometry. Based on the analysis of OSCC-related chips of GSE146483 and GSE85195 in the GEO database, it was found that hnRNP A2/B1 was differentially expressed in the OSCC and normal oral mucosa cells and tissues (all P<0.01). Meanwhile, the analysis of TSCC related chip GSE4676 confirmed that the expression of hnRNP A2/B1 was negatively correlated with the disease-free survival of TSCC patients (P=0.006). The results of real-time RT-PCR and Western blotting showed that the relative expression levels of hnRNP A2/B1 mRNA and protein in TSCC tissues were significantly up-regulated compared with those in adjacent tissues (all P<0.01). The results of Western blotting showed that the expression level of hnRNP A2/B1 in Tca-8113 cells was significantly inhibited or promoted after knockdown or overexpression of hnRNP A2/B1 (all P<0.01). The results of CCK-8 and flow cytometry showed that inhibition of hnRNP A2/B1 expression in Tca-8113 cells reduced cell proliferation activity (P<0.05) and increased cell apoptic rate (P<0.01). Overexpression of hnRNP A2/B1 in Tca-8113 cells significantly increased cell proliferation (P<0.05) and decreased cell apoptosis (P<0.01). HnRNP A2/B1 is a key factor regulating the proliferation and apoptosis of TSCC cells. Inhibition of hnRNP A2/B1 expression can reduce the proliferation activity of TSCC cells and promote the apoptosis of TSCC cells.

Loss of Caveolin-1 Expression in Tumor Cells is Associated with Increased Aggressiveness and Cell Invasion in Oral Squamous Cell Carcinoma.

Changes in Caveolin-1 (CAV-1) expression are related to tumorigenesis. The aim of this study was to evaluate the role of CAV-1 in tumor progression in oral squamous cell carcinoma (SCC) tissue samples and the effect of CAV-1 silencing on two oral tongue SCC (OTSCC) cell lines (SCC-25, from a primary tumor, and HSC-3 from lymph node metastases). Mycroarray hybridization, mRNA expression, and immunohistochemistry were performed on OSCC tissue samples and corresponding non-tumoral margin tissues. The effects of CAV-1 silencing (siCAV-1) on cell viability, membrane fluidity, on the expression of epithelial to mesenchymal transition (EMT) markers and on cell migration and invasion capacity of OTSCC cell lines were evaluated. Microarray showed a greater CAV-1 expression (1.77-fold) in OSCC tumors than in non-tumoral tissues and 2.0-fold more in less aggressive OSCCs. However, significant differences in CAV-1 gene expression were not seen between tumors and non-tumoral margins nor CAV-1 with any clinicopathological parameters. CAV-1 protein was localized both in carcinoma and in spindle cells of the tumor microenvironment (TME), and CAV-1 positive TME cells were associated with smaller/more aggressive tumors, independent of the carcinoma cells' expression. Silencing of CAV-1 increased cell viability only in SCC-25 cells. It also stimulated the invasion of HSC-3 cells and increased ECAD and BCAT mRNA in these cells; however, the protein levels of the EMT markers were not affected. Decreased expression of CAV-1 by tumor cells in OSCC and an increase in the TME were associated with increased cell invasiveness and tumor aggressiveness.

Mena as a key enhancer factor of EMT to promote metastasis of human tongue squamous cell carcinoma.

The aim of this study was to investigate the effect of mammalian-enabled (Mena) on tongue squamous cell carcinoma (TSCC) metastasis and its mechanism. Immunochemistry was performed to investigate the Mena and tumor-related markers expression, and its clinicopathological characteristics in 46 TSCC specimens. TSCC cell SCC9 and Cal27 untransfected or stable transfected with Mena overexpression and small interfering RNA were used to determine the role of Mena in cell proliferation, cell migration, invasion and metastasis, and EMT-related markers in vitro, and the effect of Mena on TSCC growth and metastasis through tumor-bearing and tumor metastasis immunodeficient mice models in vivo. Immunochemistry showed that the expression of Mena was significantly correlated with lymphatic metastasis and TNM stage, E-cadherin, Vimentin, and MMP2. Mena did not affect cell proliferation and colony formation in vitro, and tumor growth in vivo. However, it promoted cell migration and invasion in vitro, and TSCC metastasis in vivo. Mena expression is associated with lymphatic metastasis and tumor stage and promotes TSCC invasion and metastasis by inducing the EMT process. Thus, Mena may be a biomarker for prognosis and targeted therapy in TSCC patients.

The Clinical and Biological Effects of Receptor Expression-Enhancing Protein 6 in Tongue Squamous Cell Carcinoma.

There are currently no effective biomarkers for the diagnosis and treatment of tongue squamous cell carcinoma (TSCC), which causes a poor 5-year overall survival rate. Thus, it is crucial to identify more effective diagnostic/prognostic biomarkers and therapeutic targets for TSCC patients. The receptor expression-enhancing protein 6 (REEP6), a transmembrane endoplasmic reticulum resident protein, controls the expression or transport of a subset of proteins or receptors. Although it was reported that REEP6 plays a role in lung and colon cancers, its clinical impact and biological role in TSCC are still unknown. The present study aimed to identify a novel effective biomarker and therapeutic target for TSCC patients. Expression levels of REEP6 in specimens from TSCC patients were determined with immunohistochemistry. Gene knockdown was used to evaluate the effects of REEP6 in cancer malignancy (colony/tumorsphere formation, cell cycle regulation, migration, drug resistance and cancer stemness) of TSCC cells. The clinical impact of REEP6 expression and gene co-expression on prognosis were analyzed in oral cancer patients including TSCC patients from The Cancer Genome Atlas database. Tumor tissues had higher levels of REEP6 compared to normal tissues in TSCC patients. Higher REEP6 expression was related to shorter disease-free survival (DFS) in oral cancer patients with poorly differentiated tumor cells. REEP6-knocked-down TSCC cells showed diminished colony/tumorsphere formation, and they also caused G1 arrest and decreased migration, drug resistance and cancer stemness. A high co-expression of REEP6/epithelial-mesenchymal transition or cancer stemness markers also resulted in poor DFS in oral cancer patients. Thus, REEP6 is involved in the malignancy of TSCC and might serve as a potential diagnostic/prognostic biomarker and therapeutic target for TSCC patients.

Rhaponticin suppresses the hypoxia-induced factor-1 alpha-mediated aggressive phenotype of tongue squamous cell carcinoma

BackgroundEmerging evidence suggests that rhaponticin, a stilbene monomeric compound isolated from North China rhubarb, has been shown to exhibit significant biological activity against tumors. However, the anticancer effects and mechanisms of rhaponticin in tongue squamous cell carcinoma (TSCC) remain elusive.ObjectiveWe investigated the changes of migration and invasion abilities and EMT progression of TSCC cells treated with different concentrations of rhaponticin under hypoxia, as well as the possible mechanisms, in order to initially explore the effects of rhaponticin on the biological characteristics of TSCC cells under hypoxia.ResultsThe number of cell migration and invasion was prominently increased, E-cadherin protein was down-regulated, and N-cadherin and HIF-1α protein expression was elevated under hypoxia. Rhaponticin intervention strikingly prevented the increased abilities of migration and invasion and EMT of TSCC cells under hypoxia. This was followed by further validation finding that rhaponticin indeed leads to reduced HIF-1α post-transcriptional activity. Mechanistically, rhaponticin may bind to aryl-hydrocarbon nuclear translocator (ARNT) domain of HIF-1α.ConclusionsRhaponticin repressed the invasion and migration abilities and EMT process of TSCC cells under a hypoxic environment in vitro by targeted suppression of HIF-1α.

Plumbagin Enhances the Anticancer Effects of PF Chemotherapy via Downregulation of the PI3K/AKT/mTOR/p70S6K Pathway in Human Tongue Squamous Cell Carcinoma.

Cisplatin plus 5-fluorouracil (PF) is used as the standard neoadjuvant chemotherapy (also called preoperative chemotherapy) in the treatment of tongue squamous cell carcinoma (TSCC). Although PF chemotherapy reduces the distant metastasis of TSCC, the five-year survival rate has not significantly improved. In recent years, components considered in traditional Chinese medicine have been researched as adjuvant drugs for radiotherapy and chemotherapy. Plumbagin (PB) is a quinone component isolated from Plumbago zeylanica L. Notably, PB demonstrates numerous anticancer properties. In order to examine the chemosensitization effect of PB on PF and its associated mechanisms, in vitro experiments using TSCC Cal27 and cisplatin (CDDP)-resistant Cal27/CDDP cells were carried out in the present study, and the results were subsequently verified using nude mice xenografts. Results of the present study demonstrated that PB enhanced the anticancer effects of PF on the proliferation, migration, and invasion of Cal27 and Cal27/CDDP cells. Cell cycle assays demonstrated that both Cal27 and Cal27/CDDP cells were arrested in the S phase following the combined treatment of PF and PB. Moreover, the PF and PB combination group induced higher levels of apoptosis in Cal27 and Cal27/CDDP cells compared with the group treated with PF alone. In addition, the results of the present study demonstrated that combined PB and PF inhibited the PI3K/AKT/mTOR/p70S6K pathway in TSCC cells. Moreover, the weight and volumes of tumors in nude mice were reduced following treatment with a combination of PF and PB. Results of the present study also demonstrated that the expression levels of Ki67 were markedly reduced in the combined treatment group compared with the group treated with PF alone. In summary, the results of the present study demonstrated that PB enhanced the PF sensitivity of TSCC through induction of S-phase arrest and apoptosis via the PI3K/AKT/mTOR/p70S6K pathway.

Oral-specific microenvironments regulate cell behavior and anticancer drug sensitivity of tongue squamous cell carcinoma

Squamous cell carcinoma (SCC) is the most major malignant tumor of the tongue. The tongue exists at the air-liquid interface and is covered with saliva. In addition, the tongue constituent cells and tongue cancer are present under fluid flow stimulation due to the abundant capillary network and contraction of muscle tissue. Therefore, replicating both cell-cell interactions (the cellular microenvironment) and the aforementioned physical microenvironment is very important for understanding the kinetics of tongue SCC. To elucidate the effects of the cellular and physical microenvironment on tongue SCC and to investigate the relationships between these factors, we developed a collagen cell disc, with double dish under a rotational culture method to generate cancer-stroma interactions and to create fluid flow stimulation. Mesenchymal cells, NIH-3T3 cells and tongue-derived fibroblasts influenced the proliferative potential. Extracellular signal-regulated kinase and p38 signaling were regulated either synergistically or independently by cellular interactions and fluid flow stimulation, depending on the SCC cell type. The cell-cell interactions and fluid flow stimulation independently, synergistically or contradictorily affected the behavior of tongue SCC. Fluid flow stimulation synergistically enhanced the antiproliferative effect of cis-diamminedichloroplatinum on tongue SCC cells, but mesenchymal cells abolished the synergistic antiproliferative effect related to fluid flow stimulation. In conclusion, a reconstructed model was established to investigate the cellular and physical microenvironments of tongue SCC in vitro. The newly established system is a promising model for the development of further regimes to treat general oral cancer.

A Novel Tongue Squamous Cell Carcinoma Cell Line Escapes from Immune Recognition due to Genetic Alterations in HLA Class I Complex.

Immune checkpoint inhibitors (ICI) have made progress in the field of anticancer treatment, but a certain number of PD-L1 negative OSCC patients still have limited benefits from ICI immuno-therapy because of primary immune evasion due to immunodeficiency. However, in existing human OSCC cell lines, cell models that can be used to study immunodeficiency have not been reported. The objective of this study was to establish a PD-L1 negative OSCC cell line, profile whether the presence of mutated genes is associated with immune deficiency, and explore its influence on the immune recognition of CD8+ T cells in vitro. Here, we established a novel tongue SCC cell line (WU-TSC-1), which escapes from immune recognition by antigen presentation defects. This cell line was from a female patient who lacked typical causative factors. The expression of PD-L1 was negative in the WU-TSC-1 primary tumor, transplanted tumor, cultured cells and lipopolysaccharide stimulation. Whole exome sequencing (WES) revealed that WU-TSC-1 harbored missense mutations, loss of copy number and structural variations in human leukocyte antigen (HLA) class I/II genes. The tumor mutation burden (TMB) score was high at 292.28. In addition, loss of heterozygosity at beta-2-microglobulin (B2M)-a component of all HLA class I complex allotypes-was detected. Compared with the commonly used OSCC cell lines, genetic alterations in HLA class I and B2M impeded the proteins' translation and inhibited the activation and killing effect of CD8+ T cells. In all, the WU-TSC-1 cell line is characterized by genetic variations and functional defects of the HLA class I complex, leading to escape from recognition by CD8+ T cells.

Microfibril-associated protein 2 is activated by POU class 2 homeobox 1 and promotes tumor growth and metastasis in tongue squamous cell carcinoma

Tongue squamous cell carcinoma (TSCC) represents the most frequent malignancy of the oral cavity, characterized by a high metastasis rate and poor prognosis. Microfibril-associated protein 2 (MFAP2), as an extracellular matrix protein, has been found to drive tumor progression. The function and underlying mechanism of MFAP2 in TSCC remain unknown. The expression levels of MFAP2 were analyzed in tissue samples from 30 TSCC patients by real time-polymerase chain reaction and western blot assays. Our results revealed that the expression of MFAP2 mRNA and protein was upregulated in TSCC tissue samples compared with that in the matched para-carcinoma tissue samples. By performing in vitro gain-of-function or loss-of-function experiments and in vivo mouse xenograft experiments, we found that overexpression of MFAP2 induced proliferation and promoted transition from G1 to S phase of TSCC cells. Stronger invasive and migratory capabilities were observed in MFAP2-overexpressing TSCC cells. In contrast, knockdown of MFAP2 exhibited anti-proliferative, apoptosis-promoting and pro-migratory roles in TSCC cells. Knockdown of MFAP2 significantly inhibited xenograft tumor growth. Mechanistically, POU class 2 homeobox 1 (POU2F1) was recruited to the region of MFAP2 promoter and upregulates the expression of MFAP2. Silencing of MFAP2 effectively blocked the proliferation, migration, and invasion of TSCC cells caused by POU2F1 overexpression. Our results indicate that the role of MFAP2 in TSCC may attribute to transcriptional regulation of POU2F1.

Hsa_circ_0000231 affects the progression of tongue squamous cell carcinoma by activating Wnt/β-catenin signaling pathway

Objective: To explore the action mechanism of hsa_circ_0000231 in the occurrence and development of tongue squamous cell carcinoma (TSCC). Methods: Tissue samples of 60 TSCC patients were examined. The patients, including 32 males and 28 females, aged from 36 to 84 years old, underwent surgery in the Affiliated Hospital of Nantong University and Affiliated Tumor Hospital of Nantong University from December 2014 to December 2017. Saliva samples were obtained from healthy volunteers (5 males and 5 females, aged from 40 to 75 years old) and 10 TSCC patients. The TSCC cell lines (CAL-27, Tca-8113 and HN-4) were used. The expression levels of hsa_circ_0000231 in 60 pairs of freshly matched TSCC and para-carcinoma tissue samples, 10 pairs of saliva samples and 3 TSCC cell lines were detected by quantitative real-time polymerase chain reaction (qRT-PCR). hsa_circ_0000231 gene interference and lentiviral transfection were constructed, hsa_circ_0000231 in TSCC cell lines CAL-27 and Tca-8113 was knocked down, and the expressions of hsa_circ_0000231 in hsa_circ_0000231 interference group (sh-circ) and no-load lentivirus group (negative control) were tested with qRT-PCR. Cells with the highest knock-down efficiency were selected for CCK-8 test, colony formation assay, transwell invasion assay and scratch assay. The expressions of EMT-related proteins including E-cadherin, snail protein, N-cadherin and vimentin and proteins related to Wnt/β-catenin signaling pathway including β-catenin, C-myc, Bcl-2, MMP-9 and Cyclin D1 were measured by western blot. After TSCC cells in the interference group were co-cultured with Wnt/β-catenin pathway activator LiCl, the expressions of above proteins were re-measured by western blot. TSCC cells in interference group and control group were subcutaneously injected into nude mice to compare the effect of hsa_circ_0000231 knockdown on the growths of the tumors grafted subcutaneously in the nude mice. Statistical analysis software 25.0 was used for data analysis, and t-test or chi-square test was used for comparison between groups. Results: hsa_circ_0000231 was highly expressed in the tissue and saliva samples of TSCC patients and cell lines CAL-27, Tca-8113 and HN-4, but lowly expressed in paired para-carcinoma tissues, saliva samples of healthy people and normal human oral keratinocytes (all P<0.05). Log-rank univariate analysis showed that hsa_circ_0000231 expression level, tumor differentiation degree and T stage were related to the survival of TSCC patients (all P<0.05). Multivariate Cox risk regression model analysis suggested that hsa_circ_0000231 expression level (χ2=5.77,P=0.016) and T stage (χ2=5.27,P=0.029) were independent factors for the poor prognosis of TSCC patients. Western blot showed the expressions of snail protein, N-cadherin and vimentin were down-regulated, but E-cadherin was up-regulated in interference group compared with control group. In interference group, the expressions of β-catenin, C-myc, Bcl-2, MMP-9 and CyclinD1 were down-regulated, which were reversed after TSCC cells were co-cultured with LiCl. The knockdown of hsa_circ_0000231 reduced the proliferation, invasion and metastasis abilities of CAL-27 and Tca-8113 cells, which were reversed after TSCC cells were co-cultured with LiCl. The growth rate and volume of the tumors grafted subcutaneously in interference group using LiCl were greater than those in negative control group. Conclusion: hsa_circ_0000231 is an independent prognostic factor of TSCC. Highly expressed hsa_circ_0000231 can promote the proliferation, invasion and metastasis of TSCC cells.