FOXM1-Mediated Regulation of Reactive Oxygen Species and Radioresistance in Oral Squamous Cell Carcinoma Cells.

Radioresistance is a major obstacle to the successful treatment of oral squamous cell carcinoma (OSCC). To help overcome this issue, we have developed clinically relevant radioresistant (CRR) cell lines generated by irradiating parental cells over time, which are useful for OSCC research. In the present study, we conducted gene expression analysis using CRR cells and their parental lines to investigate the regulation of radioresistance in OSCC cells. Based on gene expression changes over time in CRR cells and parental lines subjected to irradiation, forkhead box M1 (FOXM1) was selected for further analysis in terms of its expression in OSCC cell lines, including CRR cell lines and clinical specimens. We suppressed or upregulated the expression of FOXM1 in OSCC cell lines, including CRR cell lines, and examined radiosensitivity, DNA damage, and cell viability under various conditions. The molecular network regulating radiotolerance was also investigated, especially the redox pathway, and the radiosensitizing effect of FOXM1 inhibitors was examined as a potential therapeutic application. We found that FOXM1 was not expressed in normal human keratinocytes but was expressed in several OSCC cell lines. The expression of FOXM1 was upregulated in CRR cells compared with that detected in the parental cell lines. In a xenograft model and clinical specimens, FOXM1 expression was upregulated in cells that survived irradiation. FOXM1-specific small interfering RNA (siRNA) treatment increased radiosensitivity, whereas FOXM1 overexpression decreased radiosensitivity, and DNA damage was altered significantly under both conditions, as well as the levels of redox-related molecules and reactive oxygen species production. Treatment with the FOXM1 inhibitor thiostrepton had a radiosensitizing effect and overcame radiotolerance in CRR cells. According to these results, the FOXM1-mediated regulation of reactive oxygen species could be a novel therapeutic target for the treatment of radioresistant OSCC; thus, treatment strategies targeting this axis might overcome radioresistance in this disease.

An oncolytic virus as a promising candidate for the treatment of radioresistant oral squamous cell carcinoma.

We evaluated the usefulness of an oncolytic virus (Suratadenoturev; OBP-301) against radioresistant oral squamous cell carcinoma. We confirmed the expression of human telomerase reverse transcriptase and the coxsackievirus and adenovirus receptor in cell lines. Also, we examined the potential presence in a patient who has received existing therapy that is amenable to treatment with OBP-301. We evaluated: (1) the antitumor effects of OBP-301 alone and in combination with radiotherapy on radioresistant cell lines, (2) the molecular mechanism underlying the radiosensitizing effect and cell death increased by the combination therapy, and (3) the antitumor effect of the combination therapy in vivo using xenograft models (a radioresistant cell line-derived xenograft in mouse and a patient-derived xenograft). Human telomerase reverse transcriptase and the coxsackievirus and adenovirus receptor were expressed in all cell lines. OBP-301 decreased the proliferative activity of these cell lines in a concentration-dependent manner, and significantly enhanced the antitumor effect of irradiation. Phosphorylated STAT3 and its downstream molecules, which correlated with apoptosis and autophagy, showed significant changes in expression after treatment with OBP-301. The combination therapy exerted a significant antitumor effect versus radiotherapy alone in both xenograft models. Combination of OBP-301 with radiotherapy exerts a synergistic effect and may represent a promising treatment for radioresistant oral squamous cell carcinoma.

Naringenin potentiates anti-tumor immunity against oral cancer by inducing lymph node CD169-positive macrophage activation and cytotoxic T cell infiltration.

The CD169+ macrophages in lymph nodes are implicated in cytotoxic T lymphocyte (CTL) activation and are associated with improved prognosis in several malignancies. Here, we investigated the significance of CD169+ macrophages in oral squamous cell carcinoma (OSCC). Further, we tested the anti-tumor effects of naringenin, which has been previously shown to activate CD169+ macrophages, in a murine OSCC model. Immunohistochemical analysis for CD169 and CD8 was performed on lymph node and primary tumor specimens from 89 patients with OSCC. We also evaluated the effects of naringenin on two murine OSCC models. Increased CD169+ macrophage counts in the regional lymph nodes correlated with favorable prognosis and CD8+ cell counts within tumor sites. Additionally, naringenin suppressed tumor growth in two murine OSCC models. The mRNA levels of CD169, interleukin (IL)-12, and C-X-C motif chemokine ligand 10 (CXCL10) in lymph nodes and CTL infiltration in tumors significantly increased following naringenin administration in tumor-bearing mice. These results suggest that CD169+ macrophages in lymph nodes are involved in T cell-mediated anti-tumor immunity and could be a prognostic marker for patients with OSCC. Moreover, naringenin is a new potential agent for CD169+ macrophage activation in OSCC treatment.

Extracellular vesicles derived from radioresistant oral squamous cell carcinoma cells contribute to the acquisition of radioresistance via the miR-503-3p-BAK axis.

Despite advancements in treatments, oral squamous cell carcinoma (OSCC) has not significantly improved in prognosis or survival rate primarily due to the presence of treatment-resistant OSCC. The intercellular communication between tumour cells is a molecular mechanism involved in acquiring OSCC treatment resistance. Extracellular vesicles (EVs) and encapsulated miRNAs are important mediators of intercellular communication. Here, we focused on EVs released from clinically relevant radioresistant (CRR) OSCC cells. Additionally, we evaluated the correlation between miRNA expression in the serum samples of patients who showed resistance to radiotherapy and in EVs released from CRR OSCC cells. We found that EVs released from CRR OSCC cells conferred radioresistance to radiosensitive OSCC cells via miR-503-3p contained in EVs. This miR-503-3p inhibited BAK and impaired the caspase cascade to suppress radiation-induced apoptosis. Furthermore, OSCC cells with BAK knockdown had increased radioresistance. Additionally, the expression of circulating miR-503-3p in patients with OSCC was correlated with a poor treatment response and prognosis of radiotherapy. Our results provide new insights into the relationship between EVs and the radioresistance of OSCC and suggest that the miR-503-3p-BAK axis may be a therapeutic target and that circulating miR-503-3p is a useful prognostic biomarker in the radiotherapy of OSCC.

miR-30a attenuates drug sensitivity to 5-FU by modulating cell proliferation possibly by downregulating cyclin E2 in oral squamous cell carcinoma.

We aimed to determine the functional role of the miRNA, which affects drug sensitivity to 5-FU in oral squamous cell carcinoma (OSCC), using two types of 5-FU-resistant and parental OSCC cell lines. MiRNA microarray data showed that miR-30a was significantly upregulated in two resistant cell lines. Therefore, we investigated the effects and molecular mechanism of miR-30a on 5-FU sensitivity. Stable overexpression of miR-30a in parental OSCC cells decreased cell proliferation and attenuated drug sensitivity to 5-FU. Cell cycle analysis indicated that miR-30a overexpression increased the proportion of G1 phase cells and decreased the proportion of S phase cells. MiR-30a knockdown using siRNA reversed the effects of miR-30a overexpression. DNA microarray analysis using miR-30a-overexpressing cell lines and a TargetScan database search showed that cyclin E2 (CCNE2) is a target of miR-30a. A luciferase reporter assay confirmed that a miR-30a mimic interacted with the specific binding site in the 3' UTR of CCNE2. CCNE2 knockdown with siRNA in OSCC cells yielded decreased drug sensitivity to 5-FU, similar to miR-30a overexpressing cells. These findings suggest that miR-30a in OSCC may be a novel biomarker of 5-FU-resistant tumors, as well as a therapeutic target for combating resistance.

Re-evaluating the clinical significance of serum p53 antibody levels in patients with oral cancer in Japanese clinical practice.

TP53 gene mutations can lead to mutant p53 protein accumulation in cancer cells, thereby inducing the production of serum antip53 antibodies (Ap53Ab) in patients with various types of cancer. The aim of the present study was to re-evaluate the clinicopathological and prognostic significance of Ap53Ab using the Ap53Ab ELISA kit, approved by the Japanese Health Insurance System in 2007. Ap53Ab was measured as a tumor marker in 94 patients with oral squamous cell carcinoma (OSCC), by subjecting paraffin-embedded sections obtained from biopsy specimens to immunohistochemical analysis to confirm p53 expression. The associations among Ap53Ab status, p53 expression and clinical significance in OSCC were examined. A total of 23% of the patients were Ap53Ab-positive. Ap53Ab status was found to be significantly associated with p53 expression status in primary tumors (P=0.027), clinical T-category, pathological N-category and pathological stage (P=0.04, P=0.010 and P=0.013, respectively). Kaplan-Meier curve analysis revealed that Ap53Ab status was significantly associated with poor disease-free survival (DFS; P=0.043), and Cox regression analysis revealed that Ap53Ab status was a significant prognostic factor for DFS in patients with OSCC (hazard ratio=2.807; 95% confidence interval: 1.029-7.160; P=0.044). These results suggested that Ap53Ab measurement may reflect the p53 mutation status and an aggressive malignant phenotype, and it may serve as a useful predictive marker candidate for OSCC in clinical practice.

Compositional Shift of Oral Microbiota Following Surgical Resection of Tongue Cancer.

Salivary microbiota is considered a source of microorganisms for the respiratory and digestive tracts, and a trigger for diseases in these distant organs. Meanwhile, the microbiota on the tongue surface is thought to be a major source of salivary microbiota. Therefore, surgical resection of the tongue for definitive treatment of oral cancer could drastically change the salivary bacterial balance and virulence. Here, we investigated the shift of the salivary microbiota following surgical resection in patients with tongue cancer. The stimulated saliva samples were collected from 25 tongue cancer patients pre- and post-resection of the tongue, and bacterial density and composition was determined using quantitative PCR analysis and 16S ribosomal RNA (rRNA) gene sequencing, respectively. Although no significant difference in the total bacterial density in saliva pre- and post-surgery was observed, the bacterial composition significantly differed according to the analysis of similarity. Among predominant operational taxonomic units (OTUs) with ≥1% of relative abundance, the proportions of OTUs corresponding to Streptococcus salivarius, Prevotellamelaninogenica, and Prevotellahisticola were significantly decreased following the tongue resection. On the other hand, the proportions of OTUs corresponding to Lautropiamirabilis, Neisseriaflava, Streptococcussanguinis, and Fusobacterium nucleatum, known to be inhabitants of dental plaque, were significantly increased. These results suggest that surgical resection of the tongue causes a compositional shift of the salivary microbiota, characterized by an increase in bacterial species derived from dental plaque, including periodontal pathogens. These results suggest the necessity of more careful and frequent postoperative oral care after surgical resection of tongue cancer.

Onodera's prognostic nutritional index correlates with tumor immune environment and survival in patients with oral squamous cell carcinoma undergoing chemoradiotherapy.

Pretreatment nutritional and immunological status is useful for predicting survival outcomes for various types of malignant tumors. Our objective was to determine the impact of the pretreatment Onodera's prognostic nutritional index (OPNI) on outcomes of patients who underwent definitive chemoradiotherapy for advanced oral squamous cell carcinoma (OSCC). We reviewed 47 patients treated for OSCC with definitive chemoradiotherapy (CRT) at our institution between January 2004 and December 2011. We determined the OPNI according to the following formula: 10 × serum albumin (g/dL) + 0.005 × total lymphocyte count (per µL). We determined the optimum OPNI cut-off through a receiver operating characteristic analysis. We analyzed the associations between OPNI status and various clinicopathological features and evaluated the effects of OPNI on the prognosis. We examined the relationships between OPNI and systemic inflammatory response parameters and analyzed intratumoral CD8+ T cells and their correlation with OPNI. The optimum OPNI cut-off was 42.7. A Kaplan-Meier curve analysis revealed that low OPNI was significantly associated with poor overall survival and cause-specific survival. The multivariate analysis revealed that low OPNI was independently correlated with poor 5 year overall survival and cause-specific survival. OPNI was significantly correlated with systemic inflammatory response parameters. Intratumoral CD8+ T cell counts in primary tumors were significantly lower for low OPNI than for high OPNI. The present data demonstrate that pretreatment OPNI is a valuable independent prognostic indicator of overall and cause-specific survival in advanced OSCC following definitive CRT. OPNI might reflect the tumor immune microenvironment characterization in OSCC.

BRD4 promotes metastatic potential in oral squamous cell carcinoma through the epigenetic regulation of the MMP2 gene.

BACKGROUND: Oral squamous cell carcinoma (OSCC) has increased morbidity, and its high metastatic potential affects patient survival. Bromodomain containing 4 (BRD4) is a chromatin protein that associates with acetylated histone lysines and facilitates transcription. BRD4 has been implicated in cell proliferation, metastasis, and prognosis in several types of cancer. However, the role of BRD4 in OSCC remains to be elucidated. METHODS: We investigated the role of BRD4 and its potential utility as a therapeutic target in OSCC. RESULTS: JQ1, the BRD4 inhibitor, suppressed the cell proliferation, migration, and invasion in the OSCC cell lines and in vivo. JQ1 reduced the expression levels of 15 metastasis genes in OSCC, including matrix metallopeptidase 2 (MMP2). Our chromatin immunoprecipitation assay showed that JQ1 reduced the BRD4 binding to the histone H3 lysine 27 acetylation-enriched sites in the MMP2 locus. Analyses of biopsy specimens from OSCC patients revealed that the BRD4 and MMP2 expression levels were correlated in the cancerous regions, and both were highly expressed in lymph node metastasis cases, including delayed metastasis. CONCLUSIONS: BRD4 contributes to metastasis in OSCC, through the epigenetic regulation of the MMP2 gene, and thus BRD4 may represent a therapeutic target and a novel prediction indicator for metastasis.

Enhanced Expression of IGFBP-3 Reduces Radiosensitivity and Is Associated with Poor Prognosis in Oral Squamous Cell Carcinoma.

Insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) modulates various cell functions through IGF-dependent or independent mechanisms. However, its biological roles in the radiosensitivity of oral squamous cell carcinoma (OSCC) remain largely unknown. The purpose of this study was to determine the clinical significance and molecular mechanisms of the association between IGFBP-3 and OSCC radiosensitivity. We performed an immunohistochemical analysis of IGFBP-3 in 52 OSCC specimens from patients treated with preoperative chemoradiotherapy and surgery (phase II study). Associations between IGFBP-3 expression and clinicopathological features were also evaluated. In addition, we examined the effects of IGFBP-3 on post-X-ray irradiation radiosensitivity and DNA damage in vitro. High IGFBP-3 expression was significantly correlated with poor chemoradiotherapy responses and prognosis. With IGFBP-3 knockdown, irradiated OSCC cells exhibited significantly higher radiosensitivity compared with that of control cells. Moreover, IGFBP-3 depletion in OSCC cells reduced phosphorylation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which is required for DNA double-strand break repair during non-homologous end joining. These findings indicate that IGFBP-3 may have a significant role in regulating DNA repair and is be a potential biomarker for predicting clinical response to radiotherapy and prognosis in OSCC.

The effectiveness of rebamipide mouthwash therapy for radiotherapy and chemoradiotherapy-induced oral mucositis in patients with head and neck cancer: a systematic review and meta-analysis.

BACKGROUND: Oral mucositis is a frequent and severe adverse event in patients undergoing chemoradiotherapy for head and neck cancers, especially grade 3 or 4 mucositis. Occurrence may result in drop-out from treatment, thereby reducing survival. We aimed to clarify the effectiveness and safety of rebamipide mouthwash for oral mucositis in patients with head and neck cancer receiving treatment. METHODS: We carried out a systematic review and meta-analysis of patients with head and neck cancer who were treated with rebamipide mouthwash. We searched PubMed, EMBASE, and Cochrane Central Register of Controlled Trials (CENTRAL), and the World Health Organization (WHO) International Clinical Trial Registry Platform. The primary outcome was the incidence of severe oral mucositis, and secondary outcomes were time from treatment start to onset of oral mucositis, the response rate of radiotherapy, and any adverse events. RESULTS: We included three studies comparing rebamipide versus placebo, all of which evaluating chemoradiotherapy induced oral mucositis. The chemotherapeutic agent was docetaxel in one study and cisplatin in the remaining two. Radiotherapy in each study consisted of 3D-conformal radiation therapy, intensity modulated radiation therapy and conventional radiation therapy, respectively. The calculated odds ratio was 0.29 [95% confidence interval (CI): 0.15 to 0.55], showing a positive association in the three studies between the incidence of grade 3-4 oral mucositis and chemotherapy for head and neck cancer. One study reported an onset of oral mucositis and the time to onset was 14.6 ± 6.4 days for the rebamipide group and 11.2 ± 4.4 days for placebo. One study reported a complete response of 8.3% for placebo and 16.7% for the rebamipide the group, and the partial response was 91.7 and 75.0%, respectively. Adverse events were reported in two studies to be 6.1 and 11.6% for placebo, and 19.4 and 26.0% in the rebamipide group, respectively. CONCLUSIONS: Rebamipide mouthwash is effective in the prevention of severe mucositis and stomatitis. However, evaluation of adverse events in observational studies are needed.

Tumor budding as a novel predictor of occult metastasis in cT2N0 tongue squamous cell carcinoma.

Occult neck metastasis is an important prognostic factor in patients with tongue squamous cell carcinoma (TSCC) who are deemed clinically negative for neck metastasis. The purpose of this study was to identify predictive factors for occult neck metastasis arising from TSCC and to determine patient prognosis. Ninety-seven patients with cT2N0 TSCC who underwent surgical resection of their primary lesion as initial therapy were enrolled in this retrospective study. Cutoff values for depth of invasion (≥3.3 mm) and the tumor budding score (≥4) were determined using receiver operator characteristic analyses. Univariate and multivariate analyses revealed that a tumor budding score ≥4 is a significant independent predictive factor for the occurrence of occult neck metastasis, which in turn is a significant independent prognostic factor. When evaluating tumor budding, we demonstrated greater interobserver and intraobserver agreement when using immunohistochemical staining for cytokeratin AE1/AE3 than with hematoxylin and eosin staining (HE). We conclude that the evaluation of tumor budding is effective for identifying populations at high risk of occult neck metastasis, which will enable the planning of appropriate therapeutic strategies for patients with cT2N0 TSCC. Furthermore, cytokeratin staining is recommended over HE staining for simpler and more accurate evaluation of tumor budding.