Kotaro Ishii

A novel function of CD82/KAI-1 on E-cadherin-mediated homophilic cellular adhesion of cancer cells.

In this study, we analyzed the effect of the metastasis suppressor CD82/KAI-1, a member of the tetraspanin superfamily, on intercellular adhesion on cancer cells. The newly established invasion assay and the cell aggregation assay revealed that CD82 strengthens E-cadherin-mediated intercellular adhesion. Interestingly, ectopic expression of CD82 stabilized E-cadherin/beta-catenin complex formation. Furthermore, CD82 reduced tyrosine phosphorylation of beta-catenin on HGF stimulation. Taken together, CD82 may stabilize or strengthen E-cadherin-dependent intercellular adhesion by regulating beta-catenin-mediated signal transduction on cancer cells, and consequently, prevent cancer cells from seceding from the primary tumor site.

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.

The T-box transcription factor Brachyury regulates epithelial–mesenchymal transition in association with cancer stem-like cells in adenoid cystic carcinoma cells

BackgroundThe high frequencies of recurrence and distant metastasis of adenoid cystic carcinoma (AdCC) emphasize the need to better understand the biological factors associated with these outcomes. To analyze the mechanisms of AdCC metastasis, we established the green fluorescence protein (GFP)-transfected subline ACCS-GFP from the AdCC parental cell line and the metastatic ACCS-M GFP line from an in vivo metastasis model.MethodsUsing these cell lines, we investigated the involvement of the epithelial–mesenchymal transition (EMT) and cancer stem cell (CSCs) in AdCC metastasis by real-time RT-PCR for EMT related genes and stem cell markers. Characteristics of CSCs were also analyzed by sphere-forming ability and tumorigenicity. Short hairpin RNA (shRNA) silencing of target gene was also performed.ResultsACCS-M GFP demonstrated characteristics of EMT and additionally displayed sphere-forming ability and high expression of EMT-related genes (Snail, Twist1, Twist2, Slug, zinc finger E-box binding homeobox 1 and 2 [Zeb1 and Zeb2], glycogen synthase kinase 3 beta [Gsk3β and transforming growth factor beta 2 [Tgf-β2]), stem cell markers (Nodal, Lefty, Oct-4, Pax6, Rex1, and Nanog), and differentiation markers (sex determining region Y [Sox2], Brachyury, and alpha fetoprotein [Afp]). These observations suggest that ACCS-M GFP shows the characteristics of CSCs and CSCs may be involved in the EMT of AdCC. Surprisingly, shRNA silencing of the T-box transcription factor Brachyury (also a differentiation marker) resulted in downregulation of the EMT and stem cell markers. In addition, sphere-forming ability, EMT characteristics, and tumorigenicity were simultaneously lost. Brachyury expression in clinical samples of AdCC was extremely high and closely related to EMT. This finding suggests that regulation of EMT by Brachyury in clinical AdCC may parallel that observed in vitro in this study.ConclusionsThe use of a single cell line is a limitation of this study. However, parallel data from in vitro and clinical samples suggest the possibility that EMT is directly linked to CSCs and that Brachyury is a regulator of EMT and CSCs.

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 downregulation 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.

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 β-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.

An Adenoid Cystic Carcinoma Cell Line Possessing High Metastatic Activity has High NF-κB Activation in Response to TNF-α

Abstract Adenoid cystic carcinoma (AdCC)is characterized by frequent recurrence and distant metastasis. Although lung metastasis in AdCC is very common, the mechanism by which this occurs is uncertain. When five AdCC cell lines(ACCS, ACCT, ACCH, Acc-3, and Acc-M) were screened for metastatic ability by injecting tumor cells into nude mice via the tail vein, lung metastases were found in mice injected with Acc-M (15/16 mice) but not in mice injected with any of the other four cell lines (0/10 mice with each line). To determine why Acc-M metastasizes to the lung but the others do not, we examined the biological characteristics of Acc-M and compared them with those of the other lines. Nuclear factor-κB (NF-κB) may play a key role in malignant tumor behaviors such as invasion and metastasis. Thus, we examined these cell lines for response to tumor necrosis factor (TNF-α), one of the typical stimulators of NF-κB. Although treatment with TNF-α stimulated matrix metalloprotease 9 (MMP-9) expression in all cell lines, the response to TNF-α varied between cell lines; the greatest stimulation was observed in Acc-M. Acc-M expressed higher levels of TNF receptors (both TNF-R1 and TNF-R2) than did the other AdCC lines. Judging from inhibitor-κBα degradation and nuclear translocation and DNA binding by NF-κB, the degree of activation of NF-κB in response to TNF-α in Acc-M cell lines was very high compared to the other lines. Moreover, the ability of Acc-M cells to adhere to endothelial cells, which was greater than that of the other cell lines, was further enhanced by pretreatment with TNF-α. Acc-M cells also expressed higher levels of sialyl Lewis x than did the other AdCC cell lines. These findings suggest that lung metastasis is mediated by tumor-endothelial cell interaction, which is probably associated with the NF-κB activation pathway. Further experiments are required to identify the molecules that mediate both lung metastasis and NF-κB activation.

Expression of Urokinase-type Plasminogen Activator (uPA), uPA Receptor, and Plasminogen Activator Inhibitor-1 in Oral Squamous Cell Carcinoma

Abstract We investigated whether the expression levels of urokinase-type plasminogen activator (uPA), uPA receptor (uPAR), and plasminogen activator inhibitor-1 (PAI-1) correlate with clinicopathological features of oral squamous cell carcinoma (SCC). We immunohistochemically examined the expression levels of uPA, uPAR, and PAI-1 in 160 biopsy specimens of oral SCC. Positive stainings for uPA, uPAR, and PAI-1 were observed mainly in SCC cells, and their intensity and number of positive cells were related to lymph node involvement ( p p p p p p p p