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

Abstract Background The 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. Methods Using 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. Results ACCS-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. Conclusions The 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.</p

miR-203 Inhibits Frizzled-2 Expression via CD82/KAI1 Expression in Human Lung Carcinoma Cells.

CD82/KAI1, a member of the tetraspanin superfamily, is a suppressor of metastasis and CD82 inhibits canonical Wnt signaling via downregulation of several Frizzled (FZD) isoforms, resulting in accumulation of β-catenin at the cell membrane. In this study, we investigated the mechanism through which CD82 inhibited FZD expression by examining the effects of microRNAs (miRNAs). The miRanda algorithm predicted 11 miRNAs from FZD sequences. Among these miRNAs, CD82 caused upregulation of miR-203 (by 2.095-fold) and downregulation of miR-338-3p (by 0.354-fold) as compared with control cells. Transfection with miR-203 and miR338-3p mimics or inhibitors revealed that miR-203 downregulated FZD2 mRNA (by 0.268-fold) and protein expression (by 0.701-fold). Moreover, transfection with the miR-203 mimic also inhibited cell migration. Therefore, these findings suggested that CD82 enhanced the expression of miR-203 and directly downregulate FZD2 expression, suppressing cancer metastasis by inhibition of the Wnt signaling pathway

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

Effects of <i>miR-203</i> and <i>miR-338-3p</i> transfection on the migration of h1299 cells.

<p>(A–F) Cell migration was evaluated using wound healing assays, as described in the Materials and Methods. Randomly chosen wound fields were photographed every 8 h for 24 h. (G, H) Wound areas were evaluated using the following formula: wound area (%) = wound area after the indicated period × 100 / initial wound area. (I) Migration ability was evaluated based on the wound area. The experiments were performed in triplicate, and the data were calculated as means ± SDs. The statistical significance of differences was analyzed using the Student’s t-test. *<i>P</i> < 0.01, **<i>P</i> < 0.05.</p

<i>miR-203</i> targeted the <i>FZD2</i> gene.

<p>The FZD2 3′-UTR sequence and complementary <i>miR-203</i>-binding sequences are shown.</p

Predicted miRNAs and target FZDs.

<p>Predicted miRNAs and target FZDs.</p

Effects of <i>miR-338-3p</i> on the expression of Frizzled (FZD) proteins.

<p>h1299 cells (h1299/zeo, h1299/CD82) were transfected with <i>miR-338-3p</i> mimic, <i>miR-338-3p</i> inhibitor, or miR controls. At 48 h after transfection, total protein was extracted and analyzed by immunoblot analysis with anti-FZD antibodies. The same blots were stripped and reprobed forβ-actin as a loading control. Experiments were repeated three times, and the most representative data are shown.</p

Expression of 11 miRNAs in h1299 cells.

<p>Total RNA was isolated from h1299 cells, and the 11 predicted miRNA levels were analyzed by real-time RT-PCR. <i>RNU6B</i> was used as an internal reference gene. Experiments were performed in triplicate, and relative miRNA levels (zeo = 1) were averaged. Asterisks indicate statistically significant differences (*<i>p</i> < 0.05, **<i>p</i> < 0.01) between the two values. Data are presented as the means ± SDs.</p

Effects of <i>miR-338-3p</i> and <i>miR-203</i> on the expression of <i>FZD</i> mRNA.

<p>h1299 cells were transfected with miRNA mimics or miRNA hairpin inhibitors, and total RNA was isolated after 48 h. mRNA levels were then analyzed by real-time RT-PCR. β-Actin was used as an internal reference gene. Experiments were performed in triplicate, and relative miRNA levels (zeo/control inhibitor = 1) were averaged. Data are presented as the means ± SDs.</p