Concomitant Loss of p120-Catenin and β-Catenin Membrane Expression and Oral Carcinoma Progression with E-Cadherin Reduction

<div><p>The binding of p120-catenin and β-catenin to the cytoplasmic domain of E-cadherin establishes epithelial cell-cell adhesion. Reduction and loss of catenin expression degrades E-cadherin-mediated carcinoma cell-cell adhesion and causes carcinomas to progress into aggressive states. Since both catenins are differentially regulated and play distinct roles when they dissociate from E-cadherin, evaluation of their expression, subcellular localization and the correlation with E-cadherin expression are important subjects. However, the same analyses are not readily performed on squamous cell carcinomas in which E-cadherin expression determines the disease progression. In the present study, we examined expression and subcellular localization of p120-catenin and β-catenin in oral carcinomas (<i>n</i> = 67) and its implications in the carcinoma progression and E-cadherin expression using immunohitochemistry. At the invasive front, catenin-membrane-positive carcinoma cells were decreased in the dedifferentiated (p120-catenin, <i>P</i> < 0.05; β-catenin, <i>P</i> < 0.05) and invasive carcinomas (p120-catenin, <i>P</i> < 0.01; β-catenin, <i>P</i> < 0.05) and with the E-cadherin staining (p120-catenin, <i>P</i> < 0.01; β-catenin, <i>P</i> < 0.01). Carcinoma cells with β-catenin cytoplasmic and/or nuclear staining were increased at the invasive front compared to the center of tumors (<i>P</i> < 0.01). Although the p120-catenin isoform shift from three to one associates with carcinoma progression, it was not observed after TGF-β, EGF or TNF-α treatments. The total amount of p120-catenin expression was decreased upon co-treatment of TGF-β with EGF or TNF-α. The above data indicate that catenin membrane staining is a primary determinant for E-cadherin-mediated cell-cell adhesion and progression of oral carcinomas. Furthermore, it suggests that loss of p120-catenin expression and cytoplasmic localization of β-catenin fine-tune the carcinoma progression.</p> </div

Correlation of percentage of catenin- and E-cadherin-positive carcinoma cells.

<p>A: The percentage of β-catenin-membrane-positive and –cytoplasm-positive at the invasive front were inversely correlated (<i>P</i> < 0.01, R² = 0.11). B: The positive correlation of the percentage of membrane-positive carcinoma cells for p120-catenin and β-catenin at the invasive front (<i>P</i> < 0.01, R² = 0.33). C: The percentage of membrane-positive carcinoma cells for p120-catenin and E-cadherin at the invasive front showed the positive correlation (<i>P</i> < 0.01, R² = 0.60). Cluster analysis grouped carcinomas into p120^high/E-cadherin^high (green) and p120^low/E-cadherin^low (red) fractions. D: The positive correlation between the percentage of membrane-positive carcinoma cells for β-catenin and E-cadherin at the invasive front (<i>P</i> < 0.01, R² = 0.29) was observed. Carcinoma cells were categorized into β-catenin^high/E-cadherin^high (green) and β-catenin^low/E-cadherin^low (red) fractions.</p

Localization of p120-catenin and β-catenin in oral normal epithelium.

<p>Expression and localization of p120-catenin (A, B) and β-catenin (D, E) in oral normal epithelium was examined by immunostaining. Arrowheads indicate the basal cells. D: negative control. Bar = 65 µm (A, C, E), and 4.3 µm (B, D).</p

Expression of p120-catenin and β-catenin in oral carcinoma cells and normal keratinocytes.

<p>Membrane binding fraction and membrane non-binding fraction of oral carcinoma cells (TSU, KOSC2, KOSC3, Ca9.22, HSC2, OSC19, SCCKN) and normal keratinocytes (HaCaT) were subjected to the immunoblot for p120-catenin (isoform 1-4) and β-catenin. Na/K ATPase and β-actin were probed as the control of membrane binding protein and membrane non-binding protein, respectively.</p

Effects of TGF-β, EGF and TNF-α on expression of catenins.

<p>Oral carcinoma cells (TSU, KOSC2, HSC2, SCCKN) and HaCaT cells were treated with TGF-β (lane b), TNF-α (lane c), EGF (lane d), TGF-β/TNF-α (lane e) or TGF-β/EGF (lane f), and subjected to immunoblot for p120-catenin and β-catenin. PBS was used to treat cells as a control (lane a). β-actin was used for the internal control.</p

Progression of Oral Squamous Cell Carcinoma Accompanied with Reduced E-Cadherin Expression but Not Cadherin Switch

<div><p>The cadherin switch from E-cadherin to N-cadherin is considered as a hallmark of the epithelial-mesenchymal transition and progression of carcinomas. Although it enhances aggressive behaviors of adenocarcinoma cells, the significance and role of cadherin switch in squamous cell carcinomas (SCCs) are largely controversial. In the present study, we immunohistochemically examined expression of E-cadherin and N-cadherin in oral SCCs (<em>n</em> = 63) and its implications for the disease progression. The E-cadherin-positive carcinoma cells were rapidly decreased at the invasive front. The percentage of carcinoma cells stained E-cadherin at the cell membrane was reduced in parallel with tumor dedifferentiation (<em>P</em><0.01) and enhanced invasion (<em>P</em><0.01). In contrast, N-cadherin-positive cells were very limited and did not correlate with the clinicopathological parameters. Mouse tongue tumors xenotransplantated oral SCC cell lines expressing both cadherins <em>in vitro</em> reproduced the reduction of E-cadherin-positive carcinoma cells at the invasive front and the negligible expression of N-cadherin. These results demonstrate that the reduction of E-cadherin-mediated carcinoma cell-cell adhesion at the invasive front, but not the cadherin switch, is an important determinant for oral SCC progression, and suggest that the environments surrounding carcinoma cells largely affect the cadherin expression.</p> </div

Localization of cadherins in normal oral epithelium and oral carcinoma tissues.

<p>A: E-cadherin was localized at basal and suprabasal cells of normal oral epithelium (a). N-cadherin-positive cells were not existed in the epithelium (b). Negative control using non-immune mouse IgG instead of primary antibody (c). <i>Bar</i> = 50 µm. B: Carcinomas at the center of tumor (a, c, e) and the invasive front (b, d, f) were stained by anti-E-cadherin (a, c) and anti-N-cadherin (b, d) antibodies. Cadherins were stained at cell membrane (arrows) or cytoplasm (arrowheads). Insets; high power view of cells pointed by an arrow (a) and arrowheads (b, d). e,f: negative control. <i>Bar</i> = 12.5 µm, and 4.3 µm (insets).</p

<i>In vitro</i> and <i>in vivo</i> cadherin expression in oral carcinoma cell lines.

<p>A: <i>CDH1</i> and <i>CDH2</i> expression were quantitatively examined by the real-time PCR. Relative expression was standardized by the expression level of <i>ß-actin</i> in each sample and calibrated with the expression in HaCaT cells. B: Oral carcinoma cells (KOSC2, HSC2 and OSC19) cultured on glass slides were stained with antibodies against E-cadherin or N-cadherin. <i>Bar</i> = 10 µm. C: Oral carcinoma cells were transplanted into the mouse tongue, and subjected to the cadherin immunostaining. Arrows indicate carcinoma cells at the peripheries of tumor cell nests; Arrowheads indicate carcinoma cells at the invasive front; Double arrowheads indicate N-cadherin-positive carcinoma cells. <i>Bar</i> = 12.5 µm.</p

Reactivity of anti-cadherin antibodies.

<p>Reactivity of anti-E-cadherin and anti-N-cadherin antibodies was examined by the immunoblot. Antibodies against E-cadherin (a, Santa Cruz Biotechnology; b, R&D System) and N-cadherin (c, Invitrogen; d, Takara; e, LifeSpan Biosciences) were used. Arrows indicate a 125 kDa band and arrowheads a 120 kDa band. Antibody b and d were used for further experiments in this study.</p

Additional file 1: Figure S1. of KrĂźppel-like factor 4 expression in oral carcinoma cells and hypermethylation at the gene promoter

Methylation susceptible sites at KLF4 gene and the promoter. DNA sequence analyzed in this study is shown (the sequence in exon 1 is capitalized). Cytosines potentially susceptible to methylation and their numerical number were highlighted in red, and a hypermethylated 237-bps region was underlined. Table S1. A summary of methylation states at each methylation susceptible cytosine. (PDF 160 kb