Oral Tongue Squamous Cell Carcinoma Cell Lines Research Articles

ADCY6 is a potential prognostic biomarker and suppresses OTSCC progression via Hippo signaling pathway.

Oral tongue squamous cell carcinoma (OTSCC) is a malignant tumor. Recently, studies have found that adenylate cyclase 6 (ADCY6) plays a pivotal role in many lethal tumors formation processes. The role of ADCY6 in OTSCC remains unknown. The expression of ADCY6 in OTSCC tissue samples was detected. The clinical significance of ADCY6 in OTSCC was analyzed by statistical methods. OTSCC cell lines were selected to analyze the biological function of ADCY6. Meanwhile, the effect of ADCY6 on the growth of OTSCC in vivo was explored using subcutaneous tumorigenesis assay. WB assay was used to detect the underlying signaling pathway. Cell function recovery test used to investigate the mechanism of ADCY6-promoting OTSCC malignant biological behavior via Hippo signaling pathway. We report that ADCY6 was obviously downregulated in OTSCC tissue samples and cell lines. Importantly, lower expression of ADCY6 indicates a poorer prognosis in patients with OTSCC, and its expression is significantly correlated with TNM stage and tumor size. Functionally, forced expression of ADCY6 can significantly inhibit the proliferation, migration, invasion, and promote apoptosis of OTSCC cells. Mechanistically, we demonstrated that ADCY6 upregulation impaired Hippo signaling pathway to reduce the malignant biological behavior of OTSCC. Generally, our findings suggest that ADCY6 suppressed Hippo signaling pathway to regulate malignant biological behavior in OTSCC, which provide new cues for further exploring the mechanism of occurrence and development of OTSCC.

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.

Detection of herpes simplex virus in oral tongue squamous cell carcinoma.

Introduction: Oral tongue squamous cell carcinoma (OTSCC) is the most common cancer of the oral cavity. Contradictory results have been observed on the involvement of herpes simplex virus 1 (HSV-1) in oral squamous cell carcinomas. Here, we aimed to study the predominance of HSV-1 or HSV-2 in oral HSV infections and to investigate the presence of HSV-1 in OTSCC and its effect on carcinoma cell viability and invasion. Methods: The distribution of HSV types one and two in diagnostic samples taken from suspected oral HSV infections was determined from the Helsinki University Hospital Laboratory database. We then analysed 67 OTSCC samples for HSV-1 infection using immunohistochemical staining. We further tested the effects of HSV-1 using six concentrations (0.00001-1.0 multiplicity of infection [MOI]) on viability and two concentrations (0.001 and 0.1 MOI) on invasion of highly invasive metastatic HSC-3 and less invasive primary SCC-25 OTSCC cell lines using MTT and Myogel-coated Transwell invasion assays. Results: Altogether 321 oropharyngeal samples were diagnosed positive for HSV during the study period. HSV-1 was the predominant (97.8%) HSV type compared with HSV-2 (detected in 2.2% of samples). HSV-1 was also detected in 24% of the OTSCC samples and had no association with patient survival or recurrence. OTSCC cells were viable even after 6days with low viral load (0.00001, 0.0001, 0.001 MOI) of HSV-1. In both cell lines, 0.001 MOI did not affect cell invasion. However, 0.1 MOI significantly reduced cell invasion in HSC-3 cells. Discussion: HSV-1 infection is predominant compared with HSV-2 in the oral cavity. HSV-1 is detected in OTSCC samples without clinical significance, and OTSCC cell survival or invasion was not affected at low doses of HSV-1.

Cholesterol depletion affects caveolin-1 expression, migration and invasion of oral tongue squamous cell carcinoma cell lines

IntroductionCholesterol is a key lipid molecule within cell membranes. This is especially true in cavelolas, invaginated membrane nanodomains, which present the protein caveolin-1 (CAV-1). It is important to note that this structure is involved in many cell signalling pathways. Additionally, high cholesterol is seen in different tumor types but little is known in regards to oral tongue squamous cell carcinoma (OTSCC). The aim of this study was to evaluate the influence of cholesterol depletion on primary (SCC-25) and metastatic (HSC-3) OTSCC cell lines. Materials and methodsCell membrane fluidity, cell viability, gene and protein expression of CAV-1 and of epithelial-mesenchymal transition (EMT) markers, cell migration in Myogel and invasion-myoma assay were evaluated after cholesterol depletion with methyl-β-cyclodextrin (MβCD - 7.5, 10 or 15 mM) ResultsDecreased cell viability and increased membrane fluidity of SCC-25 cells was seen with cholesterol depletion but cell viability was less affected and there was no effect on membrane fluidity in HSC-3. Cholesterol depletion also decreased CAV-1 at 6 h but increased it after 24 h.; both epithelial and mesenchymal EMT genes were upregulated after 6 h, followed by downregulation at 24 h in SCC-25. In HSC-3, CAV-1 was downregulated, and E-cadherin gene (ECAD) was upregulated at 6 h. Only the protein β-catenin in SCC-25 was affected, and cell migration of both cell lines was decreased, affecting SCC-25 more intensely. The invasive capacity within human myoma organotypic model was increased in SCC-25 and decreased in HSC-3. ConclusionCholesterol depletion affects CAV-1 and ECAD inversely. This affect also depends on cell type since the invasive capacity was augmented in primary cells while decreased in metastatic cells.

Selinexor assists vorinostat in inhibiting HDAC activity via promoting the accumulation of maspin in the nucleus of oral tongue squamous cell carcinoma cells.

Oral tongue squamous cell carcinoma (OTSCC) is the most common oral cancer with a low overall survival rate, necessitating effective treatments. This study reports the anti-OTSCC effect of vorinostat and selinexor. OTSCC cell lines SCC-4 and SCC-25 were cultured to determine the effects of vorinostat and/or selinexor on cell survival, invasion, migration, and apoptosis. The transplanted tumor model of SCC-25 in nude mice was established to observe the therapeutic effects of vorinostat and/or selinexor. Western blotting was used to determine protein expressions in tumor cells. The results showed that histone deacetylase 1 (HDAC1) and exportin 1 (XPO1) were highly expressed, while nuclear maspin was expressed at a low rate in SCC-4 and SCC-25 compared to the normal tongue tissue. In vitro, both vorinostat and selinexor effectively inhibited cell viability, invasion, and migration, promoted cell apoptosis, down-regulated HDAC1, Matrix Metalloproteinase 2 (MMP2), and B cell leukemia/lymphoma 2 (Bcl-2), and up-regulated nuclear maspin and cleaved caspase 3. In vivo, both vorinostat and selinexor inhibited the growth of SCC-25-bearing tumors, down-regulated the expression of Ki67, HDAC1, MMP2, and Bcl-2, and promoted the expression of nuclear maspin and cleaved caspase 3. The combination of these two drugs exhibited synergistic effects both in vivo and in vitro. Our evidence shows that vorinostat combined with selinexor is an effective treatment for OTSCC. The mechanism may be that selinexor promotes the accumulation of maspin in the nucleus, an endogenous HDAC1 inhibitory protein to inhibit the HDAC1 activity of vorinostat and exert a synergistic anti-OTSCC effect.

Radiation modulates expression and related activities of c-Met protein in oral tongue squamous cell carcinoma cell lines

Objectivesc-Met, a receptor tyrosine kinase, is involved in the growth, invasion and metastasis of a variety of cancers. In a set of cell lines from several solid tumors, a five-fold increase in c-Met expression after irradiation has been reported. This study aimed to assess if c-Met is likewise abundantly expressed in oral tongue squamous cell carcinoma (OTSCC) upon exposure to irradiation, followed by a Met-induced biological response.Materials and methodsSix OTSCC cell lines were exposed to gamma radiation doses of 2, 4, and 6 Gray. The changes in c-Met protein levels were assessed by western blot and flow cytometry. c-Met gene expression, cell migration, proliferation and cell cycle assays were performed as phenotypic readouts.ResultsIrradiation resulted in upregulation of c.Met in all cell lines with different time kinetics. On average the cells displayed minimal c-Met expression on their surface ranging from 5 to 30% of total protein. Abrupt downregulation of c-Met surface expression occurred one hour after radiation but recovered 48 h post-radiation. Intracellularly, the highest level of expression was found on day 5 after radiation exposure. Irradiation induced aggressive invasive potential of the cells as determined in cell migration assays, particularly in cell lines with the highest c-Met expression.ConclusionsThese results provide novel insights into both intracellular and extracellular dynamics of c-Met expression profiles upon irradiation of OTSCC cells in vitro. It might also suggest that radiation enhances cell migration, indicative of invasiveness, through c-Met up-regulation, at least for certain types of OTSCC cells.

JMJD3 suppresses tumor progression in oral tongue squamous cell carcinoma patients receiving surgical resection.

BackgroundJumonji domain-containing-3 (JMJD3) is reported to be a histone H3 lysine 27 (H3K27) demethylase and a tumor suppressor gene. The present study designed to investigate the crucial role of JMJD3 in oral tongue squamous cell carcinoma (OTSCC) patients who received surgical resection.MethodsWe enrolled a total of 156 OTSCC patients receiving surgical resection, including 73 patients (47%) with high expression of JMJD3 and 83 patients (53%) harboring low expression of JMJD3. Two OTSCC cell lines, SAS and Cal 27, were used to explore the modulation of cancer. GSK-J4, a potent inhibitor of JMJD3, was used to treat the two OTSCC cell lines. The Chi-square test was performed to examine between-group differences in categorical variables; the Kaplan–Meier method was used to investigate survival outcome in univariate analysis, and the Cox regression model was used for multivariate analysis.ResultsThe median follow-up period was 59.2 months and he five-year disease-free survival (DFS) and overall survival (OS) rates were 46.2% and 50.0%, respectively. Better five-year DFS (59% versus 35%) and five-year OS (63% versus 39%) were mentioned in patients with high expression of JMJD3 compared to those with low expression of JMJD3. High expression of JMJD3 was significantly associated with superior DFS and OS in the univariate and multivariate analyses. Following successful inhibition of JMJD3 by GSK-J4, western blotting analysis showed the decreased expression of Rb and p21.ConclusionOur study showed that high expression of JMJD3 is a good prognostic factor in OTSCC patients who underwent surgical resection.

WEE1 kinase inhibitor MK-1775 sensitizes oral tongue squamous cell carcinoma cells to radiation irrespective of TP53 status.

Oral tongue squamous cell carcinoma (OTSCC) frequently harbors non-functional p53 and depends on G2/M checkpoint mediated by WEE1. WEE1 suppression has been identified as a promising anti-tumor strategy. This study investigated the capacity of WEE1 kinase inhibitor (MK-1775) and its underlying mechanisms in enhancing radiation responses of OTSCC cells in vitro. WEE1 kinase expression and its downstream target (CDK1) were investigated in OTSCC versus normal oral tissue. A synergistic combination of MK-1775 with radiation on OTSCC cell lines with different p53 statuses was assessed by viability assay. The radio-sensitizing effects of MK-1775 on apoptosis, cell cycle, DNA damage, and mitotic entry were also determined. Irradiation enhanced CDK1 expression in all tested cell lines, though the effect was far more pronounced in p53 mutated cell lines. MK-1775 exhibited inhibitory effects against the survival of all cell lines and enhanced their response to the radiation. These effects were strongly elicited by induction of apoptosis and lethal mitosis, but less likely by abrogation of radiation-induced G2 arrest. These results demonstrate the efficacy of MK-1775 in enhancing the radiation effect on OTSCC in vitro associated with a significant apoptotic death rate, identifying WEE1 inhibitor as a potent radiosensitizer in OTSCC irrespective of p53 mutational status.

Overexpression of UTX promotes tumor progression in Oral tongue squamous cell carcinoma patients receiving surgical resection: a case control study

BackgroundUbiquitously transcribed tetratricopeptide repeat on chromosome X (UTX) has been identified as a histone 3 lysine 27 (H3K27) demethylase and acted as a tumor suppressor gene or oncogenic function. The current study was to explore the significance of UTX in oral tongue squamous cell carcinoma (OTSCC) patients who received surgical resection.MethodsA total of 148 OTSCC patients who underwent surgical resection were identified, including 64 patients (43%) with overexpression of UTX and 84 patients (57%) harboring low expression of UTX. We also used two OTSCC cell lines, SAS and Cal 27, to determine the modulation of cancer. Chi-square test was used to investigate the difference of categorical variables between the groups; survival outcome was analyzed using the Kaplan–Meier method in univariate analysis, and a Cox regression model was performed for multivariate analyses.ResultsUnivariate and multivariate analyses showed overexpression of UTX were significantly related to worse disease-free survival (P = 0.028) and overall survival (P = 0.029). The two OTSCC cell lines were treated with GSK-J4, a potent inhibitor of UTX, and transwell migration and invasion assays showed an inhibitory effect with a dose-dependent manner. In addition, western blot analyses also revealed the inhibition of cell cycle and epithelial-mesenchymal transition.ConclusionOur study suggests that UTX plays an important role in the process of OTSCC and overexpression of UTX may predict poor prognosis in OTSCC patients who received surgical resection.

Long non‑coding RNA MIR4713HG aggravates malignant behaviors in oral tongue squamous cell carcinoma via binding with microRNA let‑7c‑5p.

Oral tongue squamous cell carcinoma (OTSCC) is one of the most aggressive pathological types of head and neck squamous cell carcinoma, and presents with rapid local invasion and metastasis. The present study confirmed that the long non-coding (lnc) RNA MIR4713HG was markedly upregulated in both OTSCC tissues and cell lines and associated with poor survival. The present study performed a series of experiments to investigate the impact of MIR4713HG on OTSCC and revealed that upregulation of MIR4713HG had a crucial role in promoting cell proliferation and metastasis of OTSCC cell lines both in vitro and in vivo. By applying bioinformatics analyses, micro RNA let-7c-5p was observed to physically bind with MIR4713HG, and the knockdown of let-7c-5p could counteract the influence of MIR4713HG on OTSCC. Furthermore, the present study demonstrated that let-7c-5p performed its regulating role in OTSCC via affecting the expression level of transmembrane channel like 7 (TMC7). In conclusion, the present study demonstrated that lncRNA MIR4713HG acted as a pro-tumor factor facilitating cell proliferation and metastasis of OTSCC via affecting the let-7c-5p/TMC7 signaling pathway, which presents as a promising therapeutic target in OTSCC.

Effect of Sex Steroid Hormones on Tongue Cancer Cells In Vitro.

Tongue cancer is more common in men than in women. Yet the effects of sex steroid hormones on the behaviour of oral tongue squamous cell carcinoma (OTSCC) are not well known. Matrix metalloproteinase 8 (MMP8) is expressed in OTSCC and can degrade estrogen receptors (ERs). Western blot was used to examine the levels of ERβ in OTSCC cell lines (HSC-3 and SCC-25). We evaluated the effects of estradiol and dihydrotestosterone (DHT) on HSC-3 and SCC-25 cell migration, invasion and viability. The effect of estradiol on the invasion of MMP8-overexpressing (MMP8+) and empty vector HSC-3 cells was examined using 3D spheroid invasion assay. Both HSC-3 and SCC-25 cells expressed ERβ. In scratch assay, estradiol, but not DHT, reduced the migration and invasion of HSC-3 and SCC-25 cells. MMP8+ HSC-3 cells showed weaker invasion than empty vector cells, in line with previous reports. However, MMP8 overexpression did not alter the effect of estradiol on HSC-3 cell invasion in spheroid assay. Estradiol inhibited the migration and invasion of OTSCC cells, whereas DHT had no effect. Our data suggest that MMP8 does not modulate the effect of estradiol in OTSCC cells. However, the sex difference in OTSCC incidence might partly be due to protective actions of estradiol in epithelial cell carcinogenesis.

Fluctuating role of antimicrobial peptide hCAP18/LL‑37 in oral tongue dysplasia and carcinoma.

Oral tongue squamous cell carcinoma (OTSCC), the most common cancer in the oral cavity, is aggressive and its incidence is increasing globally. Human host defense cationic antimicrobial peptide‑18/antimicrobial peptide leucine‑leucine‑37 (hCAP18/LL‑37) plays a complex role in various types of cancers. In the present study, we characterized the effects of exogenous LL‑37 on three OTSCC cell lines and determined the expression of hCAP18/LL‑37 in oral dysplastic and OTSCC patient samples. Our data revealed that LL‑37, especially in high doses, mostly reduced the proliferation of OTSCC cells, but the effect was fluctuating. However, LL‑37 stimulated the migration and invasion of OTSCC cells. The high dose of LL‑37 also increased the amount of total epidermal growth factor receptor (EGFR) probably due to stabilization of the receptor to the plasma membrane. However, activation of EGFR downstream pathways was mostly decreased. Our immunohistochemical analysis showed that the hCAP18/LL‑37 expression was higher in normal/mild dysplasia than in moderate/severe dysplasia and OTSCC. The hCAP18/LL‑37 expression did not correlate with clinicopathological features or outcome of OTSCC patients. Our data suggest that LL‑37 has a fluctuating effect on proliferation, migration and invasion of OTSCC cells, but it does not seem to play a role in the progression of OTSCC.

Interleukin-17F Has Anti-Tumor Effects in Oral Tongue Cancer.

We recently showed that extracellular interleukin-17F (IL-17F) correlates with better disease-specific survival in oral tongue squamous cell carcinoma (OTSCC) patients. However, the underlying mechanisms of such effect remain obscure. Here, we used qRT-PCR to assess the expression of IL-17F and its receptors (IL-17RA and IL-17RC) in two OTSCC cell lines (HSC-3 and SCC-25) and in normal human oral keratinocytes (HOKs). IL-17F effects on cancer cell proliferation, migration, and invasion were studied using a live-imaging IncuCyte system, and a Caspase-3/7 reagent was used for testing apoptosis. 3D tumor spheroids were utilized to assess the impact of IL-17F on invasion with or without cancer-associated fibroblasts (CAFs). Tube-formation assays were used to examine the effects of IL-17F on angiogenesis using human umbilical vein endothelial cells (HUVEC). OTSCC cells express low levels of IL-17F, IL-17RA, and IL-17RC mRNA compared with HOKs. IL-17F inhibited cell proliferation and random migration of highly invasive HSC-3 cells. CAFs promoted OTSCC invasion in tumor spheroids, whereas IL-17F eliminated such effect. IL-17F suppressed HUVEC tube formation in a dose-dependent manner. Collectively, we suggest that IL-17F counteracts the pro-tumorigenic activity in OTSCC. Due to its downregulation in tumor cells and inhibitory activity in in vitro cancer models, targeting IL-17F or its regulatory pathways could lead to promising immunotherapeutic strategies against OTSCC.

Human β-Defensin 2 Expression in Oral Epithelium: Potential Therapeutic Targets in Oral Lichen Planus.

Human β-defensin 2 (hBD-2) is a potent antimicrobial peptide that participates in defense against invading bacteria. We recently showed that bacterial components and histamine, through histamine H4 receptor (H4R), are involved in the pathogenesis of the potentially malignant lesion, oral lichen planus (OLP). However, the underlying mechanisms remain unknown. We, therefore, investigated the role of hBD2–histamine crosstalk signaling in promoting OLP pathology. Biopsies from OLP and oral tongue squamous cell carcinoma (OTSCC) patients, and healthy controls were used. Two OTSCC cell lines and normal human oral keratinocytes (HOKs) were used. HBD-2 and other targets were mapped by immunostaining and analyzed by ImageJ2 software. The highly sensitive droplet-digital PCR technology and qRT-PCR were utilized to study the clinically derived and in vitro samples, respectively. H4R was challenged with the specific agonist HST-10 and inverse agonist ST-1007. HBD-2 was highly induced in OLP lesions. In contrast, hBD2 expression was attenuated in OTSCC tissues, while very low levels of hBD-2 messenger RNA (mRNA) were observed in OTSCC cells. Together with tumor necrosis factor-α (TNF-α), histamine upregulated hBD-2 mRNA expression in HOKs. Activation of H4R seems to modulate the expression of epithelial hBD-2. These findings suggest the involvement of hBD-2 in the pathogenesis of OLP and may, thus, be harnessed for therapeutic interventions in OLP.

Matrix metalloproteinase 9 inhibits the motility of highly aggressive HSC-3 oral squamous cell carcinoma cells

Pro-tumorigenic activities of matrix metalloproteinase (MMP) 9 have been linked to many cancers, but recently the tumour-suppressing role of MMP9 has also been elucidated. The multifaceted evidence on this subject prompted us to examine the role of MMP9 in the behaviour of oral tongue squamous cell carcinoma (OTSCC) cells. We used gelatinase-specific inhibitor, CTT2, and short hairpin (sh) RNA gene silencing to study the effects of MMP9 on proliferation, motility and invasion of an aggressive OTSCC cell line, HSC-3. We found that the migration and invasion of HSC-3 cells were increased by CTT2 and shRNA silencing of MMP9. Proliferation, in turn, was decreased by MMP9 inhibition. Furthermore, arresten-overexpressing HSC-3 cells expressed increased levels of MMP9, but exhibited decreased motility compared with controls. Interestingly, these cells restored their migratory capabilities by CTT2 inhibition of MMP9. Hence, although higher MMP9 expression could give rise to an increased tumour growth in vivo due to increased proliferation, in some circumstances, it may participate in yet unidentified molecular mechanisms that reduce the cell movement in OTSCC.

Nox4 Overexpression as a Poor Prognostic Factor in Patients with Oral Tongue Squamous Cell Carcinoma Receiving Surgical Resection.

Background: Nox4 has been reported to promote tumor progression of various types of cancer through many different pathways. The current study was designed to evaluate the prognostic significance of Nox4 in patients with oral tongue squamous cell carcinoma (OTSCC) receiving surgical resection. Methods: We retrospectively analyzed the 161 patients with OTSCC treated with surgical resection, including 81 patients with high expression of Nox4 and 80 patients with low expression of Nox4. Two OTSCC cell lines, SAS and SCC4, were used to investigate the proliferation activity. Results: The univariate and multivariable analyses showed that negative nodal metastasis and low expression of Nox4 were significantly associated with superior disease-free survival (DFS) and overall survival (OS). Western blotting analysis indicated that Nox4 was highly expressed in these two OTSCC cell lines and knockdown of Nox4 was successful by transfecting with Nox4 shRNA. In addition, these cell lines were also treated with a Nox4 inhibitor (GKT-137831) and the results showed GKT-137831 could inhibit the proliferation of OTSCC tumor cells in a dose-dependent manner. Conclusion: Our study suggests that Nox4 plays an important role in disease progression of OTSCC and Nox4 overexpression is a poor prognostic factor for patients with OTSCC who received surgical resection.

Abstract 3104: Establishment of an oral tongue squamous cell carcinoma cell line from a never-smoking patient

Abstract (Objective) The rising incidence of oral tongue squamous cell carcinoma (OTSCC) in patients who have never smoked and the paucity of knowledge of its biological behavior prompted us to develop a new cell line originating from a never-smoker. (Materials and methods) Fresh tumor tissue of keratinizing OTSCC was collected from a 44-year-old woman who had never smoked. Serum-free media with a low calcium concentration were used in cell culture, and a multifaceted approach was taken to verify and characterize the cell line, designated UCSF-OT-1109.(Results) UCSF-OT-1109 was authenticated by STR DNA fingerprint analysis, presence of an epithelial marker EpCAM, absence of human papilloma virus (HPV) DNA, and SCC-specific microscopic appearance. Sphere-forming assays and mouse xenografts supported its tumorigenic potential. Spectral karyotype (SKY) analysis revealed numerical and structural chromosomal abnormalities. Whole-exome sequencing (WES) identified 46 non-synonymous and 13 synonymous somatic single-nucleotide polymorphisms (SNPs) and one frameshift deletion in the coding regions. Specifically, mutations of CDKN2A, TP53, SPTBN5, NOTCH2, and FAM136A were found in the databases. Copy number aberration (CNA) analysis revealed that the cell line loses chromosome 3p and 9p, but lacks amplification of 3q and 11q (as does HPV-negative, smoking-unrelated OTSCC). It also exhibits four distinctive focal amplifications in chromosome 19p, containing 131 genes without SNPs. Particularly, 52 genes showed >3- to 4-fold amplification and could be potential oncogenic drivers.(Conclusion) We have successfully established a novel OTSCC cell line from a never-smoking patient. UCSF-OT-1109 is potentially a robust experimental model of OTSCC in never-smokers. Citation Format: Steven J. Wang, Saurabh Asthana, Annemieke van Zante, Adam B. Olshen, Frank McCormick, Osamu Tetsu. Establishment of an oral tongue squamous cell carcinoma cell line from a never-smoking patient [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3104.

Semaphorin-7A contributes to growth, migration and invasion of oral tongue squamous cell carcinoma through TGF-β-mediated EMT signaling pathway.

Oral tongue squamous cell carcinoma (OTSCC) is the most frequently encountered malignant epithelial tumors. Semaphorin-7A is a membrane-associated/secreted protein that plays an essential role in the migration and progression of human malignancies. We aimed to investigate the mechanisms of Semaphorin-7A in the growth and migration of OTSCC. The expressions of Semaphorin-7A in cells were tested by RT-PCR, Western blot, and Immunofluorescence, separately. The activities of OTSCC cells (HSC-3 and Tca8113) were analyzed by MTT, following treatment with Semaphorin-7A or PBS. The migration, invasion, and apoptosis of cells were also determined. The protein expressions of epithelial mesenchymal transition (EMT) pathway were analyzed by Western blot, after treated with Semaphorin-7A in vitro and in vivo. Finally, the mouse model of OTSCC was treated with antibody target for Semaphorin-7A (AntiSema-7A), Semaphorin-7A or PBS, then the tumor size was determined, and histopathological examination and western blot was applied for further confirmation. In OTSCC cells, Semaphorin-7A was highly expressed, and Semaphorin-7A promoted growth in multiple metastatic OTSCC cell lines. Further study indicated that Semaphorin-7A resulted in up-regulation of Snail, N-cadherin and Vimentin expression, and downregulating of E-cadherin. In addition, The Ets2-repressor factor (ERF) expression was down-regulated, and transforming growth factor (TGF-β)-induced EMT was promoted in OTSCC cells. Then, the proteins of collagen types I (CT-I) and fibronectin (FIB) were also up-regulated after Semaphorin-7A treatment. Furthermore, our results indicated that inhibition of Semaphorin-7A by antibody target for Semaphorin-7A (AntiSema-7A) suppressed OTSCC growth and increased survival in a mouse model of OTSCC. Histopathological examination confirmed the inhibitory effects in vivo. Semaphorin-7A promoted growth and migration of OTSCC by regulating TGF-β-induced EMT signaling pathway in OTSCC cells, which provided a new interconnection between the Semaphorin-7A and TGF-β-induced EMT signaling pathway.

Crosstalk between tongue carcinoma cells, extracellular vesicles, and immune cells in in vitro and in vivo models.

The crosstalk between immune cells, cancer cells, and extracellular vesicles (EVs) secreted by cancer cells remains poorly understood. We created three-dimensional (3D) cell culture models using human leiomyoma discs and Myogel to study the effects of immune cells on highly (HSC-3) and less (SCC-25) invasive oral tongue squamous cell carcinoma (OTSCC) cell lines. Additionally, we studied the effects of EVs isolated from these cell lines on the cytotoxicity of CD8+ T and NK cells isolated from three healthy donors. Our analysis included the effects of these EVs on innate immunity in zebrafish larvae. Activated immune cells significantly decreased the proliferation of both OTSCC cell lines and associated with a diminished invasion area of HSC-3 cells. In general, EVs from SCC-25 increased the cytotoxic activity of CD8+ T and NK cells more than those from HSC-3 cells. However, this effect varied depending on the source and the immune and cancer cell subgroups. In zebrafish, the amount of IL-13 mRNA was decreased by SCC-25 EVs. This study describes promising in vitro and in vivo models to investigate interactions between immune cells, cancer cells, and EVs.

CXC chemokine‑7 inhibits growth and migration of oral tongue squamous cell carcinoma cells, mediated by the epithelial‑mesenchymal transition signaling pathway.

Oral tongue squamous cell carcinoma (OTSCC) is the most common oral malignancy with different histopathological symptoms and etiology of tumorigenesis. Migration and invasion is the most important characteristics of OTSCC, and limits tumor therapy in clinics. The epithelial‑to‑mesenchymal transition (EMT) signaling pathway is an important process in the progress of tumor cell migration and invasion. Previous studies have indicated that C‑X‑C chemokine receptor‑7 (CXCR‑7) promotes the progression and metastasis of tumor cells, presenting a potential target molecule for cancer therapy. The present study investigated the inhibitory effects of C‑X‑C chemokine‑7 (CXC‑7) on human OTSCC cells both in vitro and in vivo. The results demonstrated that the Tca8113 human OTSCC cell line expressed higher levels of CXC‑7 mRNA compared with the hNOE human normal oral epithelial cell line. MTT assays indicated that CXC‑7 suppressed Tca8113 cell growth, and the cytotoxicity of CXC‑7 was indicated as the cell survival of the negative control group was significantly decreased compared with the blank control and hNOE cells. Migration and invasion assays revealed that CXC‑7 inhibited Tca8113 cell local expansion and distant metastasis. In addition, the results demonstrated that the extracellular signal‑regulated kinase (ERK)/protein kinase B (AKT) signaling pathway was inhibited after CXC‑7 treatment in Tca8113 cells. N‑cadherin, E‑Cadherin, Snail and Slug expression levels in the ERK/AKT signaling pathway were inhibited in Tca8113 cells after treatment with CXC‑7. It was demonstrated that important extracellular matrix proteins involved in cell migration, including Slug, collagen type I and Vimentin, were significantly downregulated by CXC‑7 treatment. In conclusion, CXC‑7 inhibited growth and migration in OTSCC cells, mediated by the EMT signaling pathway. This suggests that CXC‑7 serves an inhibitory role in OTSCC migration, implicating CXCR‑7 as a promising biomarker for chemokine receptor‑based drug development.