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

Mechanism of action of mU50 snoRNA.

<p>(A) Schematic representation of mU50 snoRNA binding to 28S rRNA. The mU50 snoRNA sequence contains two sites that are complementary to two methylation-target sites in the 28S rRNA sequence. Consensus C and D (and C’ and D’) box motifs are shown in shaded rectangles. The ribonucleotides that are located 5 bases prior to boxes D and D’ (indicated by the arrows) are methylated. (B) Methylation-sensitive primer extension assay for U50-sites in various mouse organs. The wedges across every two lanes indicate the concentration of dNTP (1 mM and 0.004 mM) in the reaction mix. The reverse transcriptase generates a stop signal one nucleotide before the 2′-O-methylated nucleotide in the presence of 0.004 mM dNTP. The upper and lower arrows on the right of the Figure indicate the Cm2613 and Gm2628 stop signals on 28S rRNA, respectively. wt: wild-type; Δ: ΔmU50_(HG-b) mice.</p

Molecular aspects of different organs in ΔmU50_(HG-b) and wild-type mice.

<p>(A) Northern blot analysis of mU50 snoRNA expression in eight organs from wild-type and ΔmU50_(HG-b) mice. <i>In vitro</i> transcribed (IVT) mU50 snoRNA was applied as a control. Detection of 5.8S rRNA was performed for the loading control. The signal intensities of selected organs are indicated in the panel on the right of the Figure. (B) Northern blot analysis of mU50 snoRNA and 5.8S rRNA expression in spleen obtained from wild-type (+/+), ΔmU50_(HG-b) (−/−), and maternally (−/+) and paternally (+/−)-inherited ΔmU50_(HG-b) heterozygotes. (C) PCR-SSCP analysis of the mU50 snoRNA variants in ΔmU50_(HG-b) mice. Genomic DNA from ΔmU50_(HG-b) mouse, which contains a single copy of each <i>mU50HG-a</i> paralog, was used as the control PCR template. gDNA: genomic DNA; v1, v2 and v3: plasmids that correspond to the mU50 snoRNAs encoded by <i>mU50HG-a(1)</i>, <i>-a(2)</i>, and <i>-a(3)</i>, respectively.</p

Differentially expressed genes between wild-type and ΔmU50_(HG-b) mouse B-lymphocytes detected by a comparative microarray analysis.

*<p>also known as <i>Col6a4</i> (collagen, type VI, alpha 4).</p><p>Genes that exhibit >1.5-fold up/down-regulation between wild-type and ΔmU50_(HG-b) mice are listed. Probes target to immunoglobulin genes are omitted from the list (see Materials and Methods; complete dataset is available on online GEO database under the accession number GSE41164).</p

Expression analysis of <i>Xlr3a</i> and <i>Dvwa</i> genes.

<p>TaqMan®-based real-time qPCR was conducted to validate <i>Xlr3a</i> (A) and <i>Dvwa</i> (B) expression in splenic B-lymphocytes and eight organs. The threshold value was normalized by the reference gene (<i>Tbc1d25</i>). Error bars = 1 S.D. for three biological replicates. *<i>P</i><0.05; WT: wild-type; ΔmU50: ΔmU50_(HG-b) mice.</p

Genomic structure of mU50 host-genes on mouse chromosome 9.

<p>(A) Structure of transcripts for the mouse mU50 host-genes. Three <i>mU50HG-a</i>, <i>mU50HG-a(1)</i>, <i>-a(2)</i> and <i>-a(3)</i>, and the <i>mU50HG-b</i> loci are indicated sequentially by the arrows from the terminus (ter) to the centromere (cen) on the mouse chromosome. The mU50 host-genes except for <i>mU50HG-a(1)</i> are encoded in antisense direction. The right column illustrates the corresponding host-gene transcripts and their intron-encoded mU50 snoRNA variants, v1, v2, and v3. <i>mU50HG-b</i> contains mU50_v1 and v2 in distinct introns. Note that <i>mU50HG-a(2)</i> provides alternative splice variants. One transcript (AK040733) has a 5′TOP sequence (shown by asterisk) at the first exon, regarding our previous finding of the 5′TOP <i>mU50HG-a</i> gene <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072105#pone.0072105-TanakaFujita1" target="_blank">[20]</a>. The other splice variant (AK136619) possess the same sequence region at the fifth exon as the <i>mU50HG-a(1)</i> and <i>-a(3)</i>. We do not show an additional splice variant of <i>mU50HG-a(2)</i> (AK007093) listed in the GenBank database because the nucleotide length of the mU50-containing intron (17 kb) exceeds the appropriate length required for efficient processing of C/D-type snoRNA (≈85 nucleotides <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072105#pone.0072105-Hirose1" target="_blank">[45]</a>). The primer sets used in qPCR analyses (Fig. 4) are also indicated. (B) Sequence of the mU50 variants, v1, v2 and v3. Conserved box sequences (C, D′, C′, and D) are indicated by gray rectangles. Two antisense elements, where the mU50 snoRNA interacts with the target rRNA, are indicated by broken lines. The single nucleotide polymorphisms among the variants are also shown by rectangles. NB: Northern blot.</p