Transfection of T-Box Transcription Factor BRACHYURY and SOX2 Synergistically Promote Self-Renewal and Invasive Phenotype in Oral Cancer Cells.

Recent studies suggest that epithelial⁻mesenchymal transition (EMT) correlates with cancer metastasis. In addition, there is growing evidence of the association of EMT with cancer stem cells (CSCs). Recently, we showed that the T-box transcription factor BRACHYURY could be a strong regulator of EMT and the CSC phenotype, which were effectively suppressed by a BRACHYURY knockdown in an adenoid cystic carcinoma cell line. In this study, we further tested whether BRACHYURY is a regulator of cancer stemness by means of forced expression of BRACHYURY in oral cancer cell lines. BRACHYURY, SOX2, or both were stably transfected into oral carcinoma cell lines. We analysed these transfectants with respect to self-renewal phenotypes using a sphere-formation assay, and we assessed the expression levels of EMT markers and stem cell markers using real-time reverse transcription-polymerase chain reaction (RT-PCR). Cell migration and invasiveness in vitro were evaluated using a wound healing assay and a tumour cell dissemination assay, respectively. Forced expression of BRACHYURY or SOX2 slightly increased expression of EMT and stem cell markers and the self-renewal phenotype. The expression levels, however, were much lower compared to those of cancer stem cell-like cells. Forced co-expression of BRACHYURY and SOX2 strongly upregulated EMT and stem cell markers and the self-renewal phenotype. Cell migration and invasiveness in vitro were also remarkably enhanced. These synergistic effects increased expression levels of FIBRONECTIN, SNAIL, SLUG, ZEB1, and TGF-ß2. In particular, the effects on FIBRONECTIN and TGF-ß2 were significant. We found that BRACHYURY and SOX2 synergistically promote cancer stemness in oral cancer cells. This finding points to the importance of gene or protein networks associated with BRACHYURY and SOX2 in the development and maintenance of the CSC phenotype.

Expression levels of SOX2, KLF4 and brachyury transcription factors are associated with metastasis and poor prognosis in oral squamous cell carcinoma.

The prognosis of oral squamous cell carcinoma (OSCC) patients is affected by tumor recurrence and metastasis, and cancer stem cells are hypothesized to be involved in these processes. Thus, the aim of the present study was to determine whether the expression levels of five stem cell-related transcription factors, sex determining region Y-box 2 (SOX2), octamer-binding transcription factor 4 (Oct4), avian myelocytomatosis viral oncogene homolog (c-Myc), Krüppel-like factor 4 (KLF4) and brachyury, are associated with metastasis and survival in OSCC. Immunohistochemistry was performed to analyze the expression of these proteins in biopsy specimens obtained from 108 OSCC patients. The results revealed that the expression of SOX2, Oct4, KLF4 and brachyury were significantly associated with lymph node metastasis (P=0.002, P=0.031, P=0.003 and P=0.007, respectively). In addition, the expression of KLF4 and brachyury were significantly associated with distant metastasis (P=0.014 and P=0.012, respectively). Furthermore, multivariate analysis revealed that SOX2 and KLF4 are predictive factors for lymph node metastasis [odds ratios (ORs), 4.526 and 4.851, respectively], and KLF4 is also a predictive factor for distant metastasis (OR, 9.607). In addition, OSCC patients with low co-expression of SOX2, KLF4 and brachyury exhibited a significantly lower disease-specific survival rate (78.6 vs. 100%; P=0.025; χ2=5.033) and disease-free survival rate (60.7 vs. 90.9%; P=0.015; χ2=5.897) when compared with OSCC patients with high co-expression of these factors. The results indicate that SOX2, KLF4 and brachyury serve important roles in tumor progression, and these transcription factors may thus represent clinically useful prognostic markers for OSCC.

A Novel Function of CD82/KAI1 in Sialyl Lewis Antigen-Mediated Adhesion of Cancer Cells: Evidence for an Anti-Metastasis Effect by Down-Regulation of Sialyl Lewis Antigens.

We have recently elucidated a novel function for CD82 in E-cadherin-mediated homocellular adhesion; due to this function, it can inhibit cancer cell dissociation from the primary cancer nest and limit metastasis. However, the effect of CD82 on selectin ligand-mediated heterocellular adhesion has not yet been elucidated. In this study, we focused on the effects of the metastasis suppressor CD82/KAI1 on heterocellular adhesion of cancer cells to the endothelium of blood vessels in order to further elucidate the function of tetraspanins. The over-expression of CD82 in cancer cells led to the inhibition of experimentally induced lung metastases in mice and significantly inhibited the adhesion of these cells to human umbilical vein epithelial cells (HUVECs) in vitro. Pre-treatment of the cells with function-perturbing antibodies against sLea/x significantly inhibited the adhesion of CD82-negative cells to HUVECs. In addition, cells over-expressing CD82 exhibited reduced expression of sLea/x compared to CD82-negative wild-type cells. Significant down-regulation of ST3 ß-galactoside α-2, 3-sialyltransferase 4 (ST3GAL4) was detected by cDNA microarray, real-time PCR, and western blotting analyses. Knockdown of ST3GAL4 on CD82-negative wild-type cells inhibited expression of sLex and reduced cell adhesion to HUVECs. We concluded that CD82 decreases sLea/x expression via the down-regulation of ST3GAL4 expression and thereby reduces the adhesion of cancer cells to blood vessels, which results in inhibition of metastasis.

Knockdown of the T-box transcription factor Brachyury increases sensitivity of adenoid cystic carcinoma cells to chemotherapy and radiation in vitro: implications for a new therapeutic principle.

Adenoid cystic carcinoma (AdCC) is highly metastatic and resistant to chemotherapy and radiotherapy. Recently, we reported that the T-box transcription factor Brachyury is a potential regulator of cancer stem cells (CSCs). Specifically, growth of CSCs was found to be controlled by Brachyury knockdown in AdCC. Since CSCs are resistant to chemotherapy and radiotherapy, this finding provides a new principle for therapies targeting CSCs. In the present study, we established that Brachyury knockdown suppresses chemoresistance and radioresistance in vitro. Brachyury was knocked down by transfecting Brachyury short hairpin RNA (shRNA) into the AdCC CSC cell line ACCS-M GFP. Brachyury knockdown significantly inhibited cell migration and invasion and suppressed chemoresistance. A quantitative PCR array of drug transporter genes revealed that knockdown of Brachyury caused down-regulation of ATP-binding cassette transporter genes. Furthermore, ACCS-M GFP radioresistance was significantly suppressed by Brachyury knockdown. Knockdown of Brachyury significantly sensitized ACCS-M GFP cells to chemoradiotherapy. This study demonstrates that Brachyury knockdown reduces invasiveness and chemoresistance and radioresistance of CSCs in vivo. Therefore, Brachyury knockdown may be a useful therapeutic tool for sensitizing CSCs to conventional chemoradiotherapy.

Biological characterization and analysis of metastasis-related genes in cell lines derived from the primary lesion and lymph node metastasis of a squamous cell carcinoma arising in the mandibular gingiva.

Controlling metastatic lesions is an important part of improving cancer prognosis, in addition to controlling the primary lesion. There have been numerous histological studies on primary and metastatic lesions, but little basic research has been performed using cell lines from primary and metastatic lesions belonging to the same patient. In this study, we successfully established a cell line derived from lower gingival carcinoma (WK2) as well as a line derived from secondary cervical lymph node metastasis (WK3F) through primary cultures of tissue from a patient with oral squamous cell carcinoma. We then investigated the biological characteristics of the cancer cell lines from these primary and metastatic lesions and analyzed metastasis-related genes. Comparison of the biological characteristics in vitro showed that WK3F had higher cell proliferation ability and shorter cell doubling time than WK2. WK3F also had increased cell migratory ability and higher invasive and self-replication abilities. Heterotransplantation into nude mice resulted in high tumor formation rates in the tongue and high metastasis rates in the cervical lymph nodes. Changes in WK2 and WK3F gene expression were then comprehensively analyzed using microarrays. Genes with increased expression in WK3F compared to WK2 were extracted when the Z-score was ≥2.0 and the ratio was ≥5.0, while genes with reduced expression in WK3F compared to WK2 were extracted when the Z-score was ≤-2.0 and the ratio was ≤0.2; differences were found in 604 genes. From these, MAGEC1 (88.0-fold), MMP-7 (18.6-fold), SNAI1 (6.6-fold), MACC1 (6.2-fold), and HTRA1 (0.012-fold) were selected as metastasis-related candidate genes. The results suggest that these molecules could be important for clarifying the mechanisms that regulate metastasis and provide new therapeutic targets for inhibiting tumor invasion.

T-box transcription factor Brachyury expression is correlated with epithelial-mesenchymal transition and lymph node metastasis in oral squamous cell carcinoma.

The prognosis of patients with oral squamous cell carcinoma (SCC) is influenced by the presence of lymph node metastasis. Epithelial-mesenchymal transition (EMT), a process that involves events that convert adherent epithelial cells into individual migratory cells that can invade the extracellular matrix, is critical for cancer progression. Recently, the T-box transcription factor Brachyury was reported to promote EMT in human carcinoma cell lines. We analyzed the relationship between EMT (assessed by staining for E-cadherin and Vimentin) and the expression of Brachyury in association with lymph node metastasis in oral SCC. Oral SCC biopsy specimens (152 cases) were examined immunohistochemically for the expression of E-cadherin, Vimentin and Brachyury. Expression of Brachyury was correlated with EMT (p=0.035) and was significantly associated with lymph node and distant metastasis (p<0.05). Logistic regression analysis showed that Brachyury and EMT were predictive factors for lymph node metastasis (odds ratio 4.390 and 5.936, respectively) and that EMT was a predictive factor for distant metastases (odds ratio 11.786). Our findings present clinical evidence for an important role of Brachyury in EMT in oral SCC, and suggest that Brachyury and EMT patterns are useful prognostic markers.

Retrospective Study of Selective Submandibular Neck Dissection versus Radical Neck Dissection for N0 or N1 Necks in Level I Patients with Oral Squamous Cell Carcinoma.

Objective. To evaluate the efficacy of selective submandibular neck dissection (SMND) in patients with oral squamous cell carcinoma (OSCC) with or without nodal metastasis. Patients. From a total of 384 patients with untreated OSCC who underwent radical excision, we identified 229 with clinically N0 necks and 68 with clinically N1 necks in level I. Main Outcome Measures. The Kaplan-Meier 5-year regional control and 5-year disease specific survival (DSS) were compared for SMND, radical neck dissection (RND), and modified radical neck dissection (MRND). Results. In clinically node-negative necks, the regional control rates were 85.2% with SMND and 83.3% with MRND (P = 0.89), and 5-year DSS rates were 86.5% and 87.0%, respectively, (P = 0.94). In clinically N1 necks, the regional control rates were 81.3% with SMND and 83.0% with RND (P = 0.72), and the DSS rates were 81.3% and 80.0%, respectively, (P = 0.94). Type of neck dissection was not significantly associated with regional control or DSS on either univariate or multivariate analysis using Cox's proportional hazard model. Conclusions. SMND can be effectively applied in elective and therapeutic management to patients with OSCC that are clinically assessed as N0 or N1 to level I of the neck.