Masayuki Taki

Down-regulation of Wnt-4 and up-regulation of Wnt-5a expression by epithelial-mesenchymal transition in human squamous carcinoma cells.

Gene expression of Wnt‐1, 2, 3, 4, 5a, 6 and 7a was analyzed by RT‐PCR in eleven squamous cell carcinoma (SCC) cell lines and compared with that in two normal oral keratinocyte strains. There appeared to be an inverse relationship between Wnt‐4 and Wnt‐5a expressions, i.e., Wnt‐4 was not expressed in HOC719‐NE, HOC313 or TSU cells, while Wnt‐5a was strongly expressed only in these cells. These cell lines showed decreased expression of E‐cadherin and elevated expression of vimentin accompanied with strong expressions of Snail and δEF1, which have been reported to be transrepressors of E‐cadherin and to trigger epithelial‐mesenchymal transition (EMT), suggesting associations of Wnt‐4 with epithelial phenotype and Wnt‐5a with mesenchymal phenotype of SCC cells. To study whether the expressions of these Wnt genes are regulated by EMT, we transfected a Snailexpression vector into A431 and OM1 cells, which express Wnt‐4 but not Wnt‐5a. The stably Snail‐overexpressing clones showed spindle morphology, increased expression of vimentin and decreased expression of E‐cadherin accompanied with augmented expression of δEF1. In these clones, down‐regulation of Wnt‐4 and up‐regulation of Wnt‐5a were clearly observed. These results indicated that Wnt‐4 and Wnt‐5a are oppositely affected by EMT, and down‐regulation of Wnt‐4 and up‐regulation of Wnt‐5a are possible markers of the malignant phenotype of human SCC.

Snail-Induced Down-Regulation of ΔNp63α Acquires Invasive Phenotype of Human Squamous Cell Carcinoma

p63 is a member of the p53 family and regulates crucial events in the formation of epithelial structures, but the role of p63 in tumor is unclear. We found that Snail-induced epithelial-to-mesenchymal transition (EMT) is accompanied by down-regulation of p63 in human squamous cell carcinomas (SCC). DeltaNp63alpha is the predominantly expressed p63 isoform in SCC cells. DeltaNp63 promoter activity required a CAAT/enhancer binding protein (C/EBP) binding element and was reduced remarkably by Snail. Down-regulation of DeltaNp63alpha and reduction of C/EBPalpha were observed in EMT phenotype cells, which exhibited invasive activity in vitro. p63 knockdown in cells enhanced invasive activity in the presence of E-cadherin. Conversely, forced expression of DeltaNp63alpha blocked invasive activity of cells with the EMT phenotype. These findings indicate that Snail down-regulates DeltaNp63alpha, leading to acquisition of the invasive phenotype by SCC. The invasive activity caused by down-regulation of DeltaNp63alpha does not require down-regulation of E-cadherin.

ΔNp63α-dependent expression of Id-3 distinctively suppresses the invasiveness of human squamous cell carcinoma

p63 is a member of the p53 family and ΔNp63α is the dominant‐expressing isoform of p63 in basal layer of normal stratified epithelium and human squamous cell carcinoma (SCC) cells. We have previously reported that down‐regulation of p63 was accompanied with epithelial‐to‐mesenchymal transition (EMT) by Snail‐expressing SCC cells, in which re‐expression of ΔNp63α diminished their invasiveness (Higashikawa K, Yoneda S, Tobiume K, Taki M, Shigeishi H, Kamata N. Snail‐induced down‐regulation of ΔNp63α acquires invasive phenotype of human squamous cell carcinoma. Cancer Res 2007;67:9207–13). In this study, we found that ΔNp63α positively regulated inhibitor of differentiation‐3 (Id‐3) expression. Id is a dominant negative regulator of E2A which is a transcriptional repressor of E‐cadherin. Enforced expression of Id‐3 was incapable of invoking E‐cadherin expression in the SCC cells with EMT phenotype, whereas it significantly impaired their invasiveness with down‐regulation of matrix‐metalloproteinase‐2 (MMP‐2) expression. Reporter gene assay revealed that the Ets‐1‐induced MMP‐2 promoter activity was suppressed by the Id‐3, while the Id‐3‐dependent E‐cadherin promoter activity was remarkably reduced in the presence of Snail. Furthermore, knockdown of p63 in SCC cells significantly decreased Id‐3 expression, in which up‐regulation of MMP‐2 expression was concomitant with the acquired invasiveness. These findings propose a particular role of the off‐signaling of the ΔNp63α‐Id‐3 axis incident to Snail‐mediated EMT for the MMP‐2‐dependent invasiveness in SCC cells.

Regulation of CXCL9/10/11 in Oral Keratinocytes and Fibroblasts

Th1 and Th2 cytokines such as interferon-γ (IFN-γ ) , tumor necrosis factor- α (TNF-α ), and IL-4 are expressed in T-cell-mediated inflammation in the oral cavity. We tested the hypothesis that those cytokines may act on CXCR3-agonistic chemokines, T-cell recruiting factors, and on neighboring cells, including oral keratinocytes and fibroblasts. Human immortalized oral keratinocytes (RT7) and fibroblasts (GT1) after 24-hour stimulation with IFN-γ showed increased mRNA levels of CXCL9 (600- and 700-fold), CXCL10 (10,000- and 150-fold), and CXCL11 (5000- and 300-fold), respectively. In contrast, TNF-α caused an increase in CXCL9 (300-fold), CXCL10 (2000-fold), and CXCL11 (2000-fold) mRNA levels in GT1, but not RT7 cells, at 24 hrs. IL-4 reinforced the promotion of CXCL9, CXCL10, and CXCL11 expression by IFN-γ in RT7 cells, whereas IL-4 inhibited the increased levels by IFN-γ and TNF-α in GT1 cells. Thus, IFN-γ , TNF-α , and IL-4 appear cooperatively to regulate CXCR3-agonistic chemokines in oral keratinocytes and fibroblasts in T-cell-mediated oral inflammation sites.

Gene expression profiling to identify genes associated with high-invasiveness in human squamous cell carcinoma with epithelial-to-mesenchymal transition

The process of epithelial-to-mesenchymal transition (EMT) involves the acquisition of high-invasiveness by tumor. Snail represses target genes and induces EMT. In this study, we defined the signatures of gene expressions by cDNA microarray analyses in both human squamous cell carcinoma (SCC) cell lines with spontaneous EMT and with Snail-induced EMT, which exhibited high-invasive behavior in vitro. Of the 17,000 cDNA probes, 61 genes were found differentially expressed with >2- or <0.5-fold ratio and shared among the EMT phenotype cell lines, indicating candidates for invasion-associated genes regulated by Snail. Category analysis showed that these genes were mainly classified as development/differentiation, metabolism, apoptosis, angiogenesis and cell adhesion. These data illustrated that Snail regulates various molecular pathways for the establishment of EMT and the acquisition of high-invasiveness in SCC cells, yielding insight into the progression of SCC.

Gene expression of telomerase related proteins in human normal oral and ectocervical epithelial cells

We analyzed telomerase activities and gene expressions of telomerase components: hTERT, hTR, hTEP1, telomeric repeat binding factors: TRF1, TRF2, and c-myc, Max and Mad in human normal oral and ectocervical epithelial keratinocytes, comparing with those of squamous carcinoma cells and HPV16- or SV40-immortalized cells. Significant telomerase activity and hTERT expression were detected in primary keratinocytes. However, both were dramatically down-regulated during serial passages. The down-regulation of hTERT mRNA was associated with augmented expression of TRF1. Expression of c-myc was slightly decreased, whereas Mad was expressed in parallel with that of hTERT during passages. We also detected an alternate splicing of hTERT transcript in two of four cancer cells and normal aged epithelial cells. These results suggest that the senescence of normal oral and ectocervical keratinocytes is accompanied with up-regulation of TRF1 and down-regulation of telomerase activity due to transcriptional suppression of active form of hTERT in vitro.

Malignant ossifying fibromyxoid tumor of the tongue: case report and review of the literature

Ossifying fibromyxoid tumor (OFMT) is a rare mesenchymal neoplasm that arises in subcutaneous tissue, with that in the oral cavity extremely rare. We present a case of malignant OFMT in the tongue. A 26-year-old male noticed a painless mass in the tongue, which was extracted at a general hospital. Four years later, the tumor recurred and was resected at our department. Histologically, the recurrent tumor was composed of the closely packed cells positive for vimentin and S-100 proliferating in a nodular fashion. It showed high cellularity and mitotic activity. In the primary tumor, some tumor cells were arranged in a diffuse or cord-like manner within an abundant fibromyxoid matrix, along with a small amount of metaplastic ossification, corresponding with the histopathological characteristic of OFMT. Accordingly, a diagnosis of malignant OFMT arising in typical OFMT was established. This is the first reported case of malignant OFMT in the tongue. Long-term follow-up is needed for confirmation of prognosis and biological behavior.

Increased Expression of CENP-H Gene in Human Salivary Gland Carcinomas

Abstract There have been very few studies on the expression of Centromere proteins in human salivary gland carcinomas. The purpose of this study was to clarify the correlation between Centromere protein H (CENP-H) expression and clinicopathologic factors in salivary gland carcinomas. The expression of CENP-H mRNA was investigated in 28 human salivary gland tumors (7 pleomorphic adenomas, 3 Warthin tumors, 6 mucoepidermoid carcinomas, 6 adenoid cystic carcinomas, 5 acinic cell carcinomas and 1 malignant myoepithelioma) and 8 normal submandibular glands using real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). The labeling index of PCNA and Ki-67 were also investigated immunohistochemically in 16 salivary gland carcinomas. The mean expression level of CENP-H mRNA was significantly higher in malignant tumors (0.55 ± 0.68) than normal submandibular glands (0.10 ± 0.029). A significant correlation between the PCNA labeling index and CENP-H mRNA expression was also found (Spearmans correlation coefficient by rank test, P=0.033). We also found a significant correlation between the Ki-67 labeling index and CENP-H mRNA expression in malignant tumors (Spearmans correlation coefficient by rank test, P=0.040). These results indicate that human CENP-H mRNA is closely linked to increased or abnormal cell proliferation in malignant salivary gland tumors.