Genta Maeda

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

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 (n = 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 (P<0.01) and enhanced invasion (P<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 in vitro 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.

Epigenetic inactivation of E-cadherin by promoter hypermethylation in oral carcinoma cells

The loss of E-cadherin expression by epigenetic aberrations, including promoter hypermethylation and transcription repressor binding, plays a key role in the initiation of the epithelial–mesenchymal transition, which leads to the progression of oral squamous cell carcinomas. However, mutual actions and roles of the epigenetic pathways remain to be elucidated. In this study, we determined the methylation status of cytosine within CpG islands of the E-cadherin promoter region in relation to the expression level of SIP1, a major E-cadherin repressor in oral carcinoma cells. Methylation-specific polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism analyses showed that the expression of E-cadherin was downregulated in parallel with promoter hypermethylation. The use of a bisulfite-modified sequence further validated that methylation was observed in 22.6 ± 38.7% (mean ± 1 SD) of cytosines in carcinoma cells negligibly expressing E-cadherin, in contrast to 7.5 ± 1.8% in E-cadherin-expressing cells. Treatment with a demethylating reagent, 5-azacytidine, induced upregulation of E-cadherin in some E-cadherin-expressing carcinoma cell lines but not in others. The finding that the unresponsive cell lines retained high expression of SIP1 supports the repressive effect of SIP1 on E-cadherin expression regardless of promoter hypermethylation. Collectively, the overall results suggest the dynamic but differential regulation of E-cadherin by epigenetic aberrations in the pathology of oral carcinomas.

Epigenetic Loss of Mucosa-Associated Lymphoid Tissue 1 Expression in Patients with Oral Carcinomas

Mucosa-associated lymphoid tissue 1 (MALT1), which is located in a genomic region that encodes unknown tumor suppressor gene(s), activates nuclear factor-kappaB in lymphocyte lineages. However, its expression and role in the pathology of malignant tumors of epithelial origin is not known. In the present study, we examined MALT1 expression and its implications for the pathology of oral carcinomas. Immunostaining localized MALT1 in the nucleus of normal oral epithelial cells, but the expression was absent in 45.0% of carcinomas (49 of 109 cases) especially at the invasive front. The loss of expression was correlated with tumor recurrence (P = 0.007) and poor patient survival (P < 0.001), and it was an independent prognostic determinant (P < 0.001). MALT1-negative carcinomas exhibited microsatellite instability at the MALT1 locus and a specific cytosine methylation positioned at -256 from the gene, and the expression was recovered by demethylation treatment. In contrast to lymphocyte lineages, carcinoma cells showed MALT1 located at the nucleus independent of its domain structures, and its loss of expression induced the epithelial-mesenchymal transition. These results show that MALT1 is expressed in the nucleus of oral epithelial cells and that its expression is epigenetically inactivated during tumor progression, suggesting that the detection of MALT1 expression is a useful predictive and prognostic determinant in the clinical management of oral carcinomas.

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

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 (n = 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, P < 0.05; β-catenin, P < 0.05) and invasive carcinomas (p120-catenin, P < 0.01; β-catenin, P < 0.05) and with the E-cadherin staining (p120-catenin, P < 0.01; β-catenin, P < 0.01). Carcinoma cells with β-catenin cytoplasmic and/or nuclear staining were increased at the invasive front compared to the center of tumors (P < 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.

Identification of GATA3 binding sites in Jurkat cells

Determining binding sites of transcription factors is important for understanding the transcriptional control of target genes. Although a transcription factor GATA3 plays a pivotal role in Th2 lymphocyte development, its physiological role is not clearly defined because the target genes remain largely unknown. In this study, we modified chromatin immunoprecipitation (ChIP), and isolated 121 GATA3 binding sites and 83 different annotated target genes. Re-ChIP analysis using anti-GATA3 and anti-RNA polymerase II mAbs and chromosome conformation capture assay demonstrate that GATA3-bound fragments interact with basal transcriptional units of target genes. GATA3 regulation of target genes under the control of binding fragments was confirmed by reporter assay and quantification of target gene mRNA expression in the presence of GATA inhibitor or short interfering RNA against GATA3. These data demonstrate that GATA3 binds to regulatory elements and controls target gene expression through physical interaction with core promoter regions.

The Role of WNT in Rheumatoid Arthritis and its Therapeutic Implication

Rheumatoid arthritis (RA) is a systematic inflammatory and intractable disease, which progressively affects multiple joints. Recent findings strongly suggest a key role of WNT signaling in the disease initiation and progression. In this review, we discuss the role and possibility of treatment by targeting WNT signaling.

Mutational analysis of HRAS and KRAS genes in oral carcinoma cell lines

RAS overexpression and its active mutations are involved in malignant tumorigenesis. However, the mutation rates in oral carcinoma cells differ between populations. In the present study, genomic DNA of oral carcinoma cells (HOC313, TSU, HSC2, HSC3, KOSC2, KOSC3, SCCKN, OSC19, Ca9.22, and Ho1u1 cells) or normal gingival fibroblasts (GF12 cells) derived from a Japanese population were amplified by polymerase chain reaction using primer sets, spanning HRAS and KRAS exons. Nucleotide substitutions were analyzed by single strand conformation polymorphism. In contrast to no substitutions in KRAS, nine different substitutions were detected in HRAS. Of the nine, six substitutions were located at intron 1 (HSC2 and HSC3 cells) or intron 2 (HSC3, SCCKN and Ca9.22 cells), and one each of exon 1 (all cells), exon 2 (HOC313, TSU, HSC2 and HSC3 cells) and the 5′ upstream region (all cells). Substitutions at exons 1 and 2 did not affect the amino acid sequence; the exon 1 substitution was positioned at the 5′ untranslated region, which may be a single nucleotide polymorphism (SNP) sequence because all the cells were isolated from a Japanese population, and the mutations at exon 2 was a silent mutation. A substitution at the 5′ upstream region was an SNP. These data demonstrate that SNPs and point mutations observed in HRAS do not change the amino acid sequence, and suggest that the mutations affecting the amino acid sequence may be a rare event in oral carcinomas of the Japanese population.

Cadherin Expression and Progression of Squamous Cell Carcinomas of the Oral Cavity

Cell-cell adhesion plays fundamental and dynamic roles in the development and maintenance of multi-cellular organisms. Epithelial sheet is a typical structure and composed of cells that work together and separate from a lumen or space from underlying tissue. It lines most internal surfaces, including gastrointestinal tract and kidney tubes, and external layer of the epithelium as the epidermis of skin. The oral cavity is covered with stratified squamous cell epithelium in which keratinizing epithelial cells strongly connect with each other and differentiate from basal cells at the bottom to keratinized surface cells. Epithelial cells are connected together by junctional complexes that have distinct order with respect to their ultra-structures; zonula occludens (tight junctions), gap junctions, zonula adherens (adherence junctions) and macula adherens (desmosomes). Adherence junctions in epithelial sheet are belt like junctions and composed of cadherins that bind with proteins at the cytoplasmic domain. In other cell types, adherence junctions display different morphology; spotty and discontinuous in fibroblastic cells and punctate in the synaptic junctions. Desmosome is a spot-like junction associated with desmosomal cadherins (desmogleins and desmocollins) and tightly associated with adjacent cell membranes compared to adherence junctions. Stratified squamous epithelial cells express large amount of cadherins and well organize adherence junctions and desmosomes. Disruption of desmosomes by autoantibodies against desmoglein causes pemphigus that are multiple and bullous diseases in the skin and oral mucosa. Cadherins are most characterized cell-cell adhesion molecules and implicated in the development and progression of carcinomas of the epithelial origin. In this chapter, we overview the regulation and role of cadherins in the pathology of oral squamous cell carcinomas (OSCCs).