PDZ-binding kinase inhibitor OTS514 suppresses the proliferation of oral squamous carcinoma cells.

OBJECTIVE: PDZ-binding kinase (PBK) has been reported as a poor prognostic factor and is a promising molecular target for anticancer therapeutics. Here, we aimed to investigate the effect of specific PBK inhibitor OTS514 on the survival of OSCC cells. METHODS: Four OSCC cell lines (HSC-2, HSC-3, SAS, and OSC-19) were used to examine the effect of OTS514 on cell survival and apoptosis. DNA microarray analysis was conducted to investigate the effect of OTS514 on gene expression in OSCC cells. Gene set enrichment analysis was performed to identify molecular signatures related to the antiproliferative effect of OTS514. RESULTS: OTS514 decreased the cell survival of OSCC cells dose-dependently, and administration of OTS514 readily suppressed the HSC-2-derived tumor growth in immunodeficient mice. Treatment with OTS514 significantly increased the number of apoptotic cells and caspase-3/7 activity. Importantly, OTS514 suppressed the expression of E2F target genes with a marked decrease in protein levels of E2F1, a transcriptional factor. Moreover, TP53 knockdown attenuated OTS514-induced apoptosis. CONCLUSION: OTS514 suppressed the proliferation of OSCC cells by downregulating the expression of E2F target genes and induced apoptosis by mediating the p53 signaling pathway. These results highlight the clinical application of PBK inhibitors in the development of molecular-targeted therapeutics against OSCC.

Inhibition of VEGFR2 and EGFR signaling cooperatively suppresses the proliferation of oral squamous cell carcinoma.

BACKGROUND: Epidermal growth factor receptor (EGFR) is frequently overexpressed in oral squamous cell carcinoma (OSCC), and EGFR-targeting therapeutics have been widely employed to treat patients with a variety of carcinomas including OSCC. Here, we aimed to investigate alternative signaling for OSCC survival under the disruption of EGFR signaling. METHODS: OSCC cell lines, namely HSC-3 and SAS, were utilized to investigate how EGFR disruption affects cell proliferation. Gene set enrichment analysis was performed to examine how EGFR disruption affects oncogenic signaling in OSCC cells. Disruption of KDR gene was performed using CRISPR/Cas9 techniques. A VEGFR inhibitor, vatalanib was used to research the impact of VEGFR inhibition on OSCC survival. RESULTS: EGFR disruption significantly decreased the proliferation and oncogenic signaling including Myc and PI3K-Akt, in OSCC cells. Chemical library screening assays revealed that VEGFR inhibitors continued to inhibit the proliferation of EGFR-deficient OSCC cells. In addition, CRISPR-mediated disruption of KDR/VEGFR2 retarded OSCC cell proliferation. Furthermore, combined erlotinib-vatalanib treatment exhibited a more potent anti-proliferative effect on OSCC cells, compared to either monotherapy. The combined therapy effectively suppressed the phosphorylation levels of Akt but not p44/42. CONCLUSION: VEGFR-mediated signaling would be an alternative signaling pathway for the survival of OSCC cells under the disruption of EGFR signaling. These results highlight the clinical application of VEGFR inhibitors in the development of multi-molecular-targeted therapeutics against OSCC.

Tumor-infiltrating FoxP3+ T cells are associated with poor prognosis in oral squamous cell carcinoma.

OBJECTIVES: Squamous cell carcinoma is the most common malignancy in the oral cavity. Moreover, human papillomavirus (HPV) infection has been recently implicated in the onset of oral squamous cell carcinoma (OSCC). Regulatory T cells (Tregs) are Forkhead box P3 (FoxP3) positive and are normally involved in the mechanism by which organisms escape attacks from their own immune system; however, in tumors, these cells are known to suppress antitumor immunity and block the attack against tumors. The present study evaluated the associations of the number of Tregs and HPV infection with prognoses in patients with OSCC. MATERIAL AND METHODS: Samples from 106 patients diagnosed with OSCC were evaluated by immunohistochemical staining for the identification of FoxP3+ Tregs and HPV. The relationship between the observed number of Foxp3-positive cells, the presence/absence of HPV infection and associations with clinicopathological indicators were analyzed. RESULTS: Tissues were classified into high (High) and low (Low) Treg count groups, with 69 patients classified as High and 37 classified as Low. The prognoses were significantly better in the Low group compared with the High group (p = 0.04). FoxP3 expression may have had some effect on nodal metastases (p = 0.09). HPV antigens were detected in 65 patients, but there were no significant associations with prognosis (p = 0.34). HPV-infected tumors were more common in the gums and tongues than in the lips, cheeks, and floor of the mouth (p = 0.05). CONCLUSIONS: These results indicate that Tregs in tumor sites are associated with worsened prognoses of patients with OSCC and suggest potential therapies targeting Tregs in OSCC.

Evaluation of skeletal muscle mass using prediction formulas at the level of the 12th thoracic vertebra.

OBJECTIVES: People with cancer have a high risk of cachexia and sarcopenia, which are associated with worse clinical outcomes. We evaluated the prediction accuracy of the Matsuyama et al. and Ishida et al. formulas using computed tomography (CT) slices from the twelfth thoracic vertebra (Th12) level in people with cancer. METHODS: This retrospective study included patients with advanced cancer who underwent thoracic and abdominal CT scans (n = 173). The cross-sectional area (CSA) on CT images was measured at the levels of Th12 and the third lumbar vertebra (L3). The Matsuyama et al. formula used the Th12 CSA, whereas the Ishida et al. formula used only the Th12 CSA of the spinal erectors; thus, the measurements were performed separately. The correlation between predicted and actual L3 CSA was assessed using r and the intraclass correlation coefficient. A prediction-accuracy analysis of the predicted values was also performed. RESULTS: The mean participant age was 66.2 ± 12.8 y; 50.3% of participants were women and 49.7% were men. Strong correlations were observed between the predicted and measured L3 values calculated from the two prediction formulas. The prediction-accuracy analysis using previously reported cutoff values showed that the Ishida et al. method had high sensitivity and the Matsuyama et al. method had high specificity for low skeletal muscle index determined by the predicted and measured L3 skeletal muscle index. CONCLUSIONS: Both the Matsuyama et al. and Ishida et al. formulas had good reliability on CT slices at the Th12 level in people with advanced cancer, indicating that these formulas can be applied in clinical practice.

Assessing skeletal muscle mass based on the cross-sectional area of muscles at the 12th thoracic vertebra level on computed tomography in patients with oral squamous cell carcinoma.

OBJECTIVES: This study aimed to create a formula to estimate the third lumbar vertebra (L3)1 level skeletal muscle cross-sectional area (CSA), known as a standard value to evaluate skeletal muscle mass on computed tomography (CT), using the twelfth thoracic vertebra (Th12) level skeletal muscle CSA on chest CT. MATERIALS AND METHODS: This retrospective observational study included patients aged 40 + years with a diagnosis of oral squamous cell carcinoma (n = 164). Skeletal muscle CSA on CT images was measured using the Th12 and the L3 levels of pretreatment CT scans. The predictive formula was created based on the five-fold cross-validation method with a linear regression model. Correlations between the predicted L3-level CSA and the actual L3-level CSA were evaluated using r and Intraclass Correlation Coefficients (ICC). RESULTS: The predictive formula for L3-level CSA from Th12-level CSA was: CSA at L3 (cm2) = 14.143 + 0.779 * CSA at Th12 (cm2) - 0.212 * Age (y) + 0.502 * Weight (kg) + 13.763 * Sex. Correlations between the predicted and measured L3-level CSA were r = 0.915 [0.886-0.937] and ICC = 0.911 [0.881-0.934]. CONCLUSION: We developed a formula for predicting skeletal muscle mass from the Th12-level CT slice. The predicted L3-level CSA correlated with the measured L3-level CSA.

Discovery of novel molecular characteristics and cellular biological properties in ameloblastoma.

Ameloblastoma is a rare odontogenic benign tumor accounting for less than 1% of head and neck tumors. Advanced next generation sequencing (NGS) analyses identified high frequency of BRAF V600E and SMO L412F mutations in ameloblastoma. Despite the existence of whole genomic sequence information from patients with ameloblastoma, entire molecular signature of and the characteristics of ameloblastoma cells are still obscure. In this study, we sought to uncover the molecular basis of ameloblastoma and to determine the cellular phenotype of ameloblastoma cells with BRAF mutations. Our comparative cDNA microarray analysis and gene set enrichment analysis (GSEA) showed that ameloblastoma exhibited a distinct gene expression pattern from the normal tissues: KRAS-responsive gene set is significantly activated in ameloblastoma. Importantly, insulin like growth factor 2 (IGF2), a member of KRAS-responsive genes, enhances the proliferation of an ameloblastoma cell line AMU-AM1 with BRAF mutation. In addition, Toll-like receptor 2 (TLR2) knockdown readily inactivated KRAS-responsive gene sets as well as increases caspase activities, suggesting that TLR2 signaling may mediate cell survival signaling in ameloblastoma cells. Collectively, the findings may help to further clarify the pathophysiology of ameloblastoma and lead to the development of precision medicine for patients with ameloblastoma.

Inhibition of Nox1 induces apoptosis by attenuating the AKT signaling pathway in oral squamous cell carcinoma cell lines.

NADPH oxidases, also known as the Nox family, are major sources of reactive oxygen species generation that regulate redox-sensitive signaling pathways. Recent studies have implicated the Nox family in cancer development and progression. However, the involvement of its members in the development of oral squamous cell carcinoma (OSCC) remains to be elucidated. To clarify this issue, we first analyzed mRNA expression of Nox/Duox family members (Nox1, Nox2, Nox3, Nox4, Nox5, Duox1 and Duox2) in five OSCC cell lines. Nox1 and Nox4 mRNAs were highly expressed in four OSCC cell lines. Western blot analysis revealed that the protein expression level of Nox1 was higher than that of Nox4 in the OSCC cell lines. In addition, knockdown of Nox1, but not Nox4, significantly suppressed cell viability and induced apoptosis in the HSC-2 and HSC-3 cells. We also found that a specific AKT inhibitor, perifosine, dose-dependently suppressed OSCC cell growth. Notably, Nox1 knockdown significantly attenuated the phosphorylation level of AKT. Furthermore, both Nox1 knockdown and perifosine treatment markedly enhanced the cisplatin-induced cytotoxic effect. Taken together, our results highlight that the Nox1/AKT signaling pathway plays an important role in cell survival in OSCC cells.