Abstractp63 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 epithelialto-mesenchymal transition (EMT) is accompanied by downregulation of p63 in human squamous cell carcinomas (SCC). #Np63A is the predominantly expressed p63 isoform in SCC cells. DNp63 promoter activity required a CAAT/enhancer binding protein (C/EBP) binding element and was reduced remarkably by Snail. Down-regulation of #Np63A and reduction of C/EBPA 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 #Np63A blocked invasive activity of cells with the EMT phenotype. These findings indicate that Snail down-regulates #Np63A, leading to acquisition of the invasive phenotype by SCC. The invasive activity caused by down-regulation of #Np63A does not require down-regulation of E-cadherin.

We have previously established immortalized cells (HCF) from cementifying fibroma of the jaw bone. Here, we found that the receptor for hyaluronan (HA)-mediated motility (RHAMM) and epiregulin, a ligand for the epidermal growth factor receptor (EGFR), were highly expressed in HCF cells in comparison with osteoblasts by conducting a microarray analysis. The cell growth of HCF cells was significantly decreased by the knockdown of RHAMM using small interfering RNA (siRNA). RHAMM was associated with extracellular signal-regulated kinase (ERK) and essential for ERK phosphorylation. HCF cells had characteristic growth mechanisms in which epiregulin functions in an extracellular autocrine loop. Interestingly, exogenous HA induced the phosphorylation of EGFR, which was mainly dependent on CD44. The results raise the novel idea that the EGFR may activate Raf-MEK-ERK signaling in response to the binding of HA to CD44. Moreover, RHAMM was able to associate with TPX2 in the nucleus and was required for HA-induced activation of the Aurora A kinase. The results suggest that RHAMM has a predominant role in the cell cycle in HCF. Here, we report the new machinery by which RHAMM/ERK interaction induces the proliferative activity of cementifying fibroma cells via a specific signaling pathway through the CD44-EGFR axis. Benign fibro-osseous diseases in the oral and maxillofacial regions can be divided into three categories: fibrous dysplasia, osseous dysplasia, and cementifying (ossifying) fibroma.

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Our results show that GSK3β plays a major role in maintenance of stemness of CD44(high) /ALDH1(high) HNSCC cells. Additionally, they indicate a close relationship between CSC and mesenchymal characteristics in HNSCC.

p63 is a member of the p53 family and DNp63a is the dominantexpressing 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 Snailexpressing SCC cells, in which re-expression of DNp63a diminished their invasiveness (Higashikawa K, Yoneda S, Tobiume K, Taki M, Shigeishi H, Kamata N. Snail-induced down-regulation of DNp63a acquires invasive phenotype of human squamous cell carcinoma. Cancer Res 2007;67:9207-13). In this study, we found that DNp63a 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 DNp63a-Id-3 axis incident to Snail-mediated EMT for the MMP-2-dependent invasiveness in SCC cells. ' UICCKey words: Id-3; p63; tumour invasion; squamous cell carcinoma; snail Epithelial-to-mesenchymal transition (EMT) is the process of disaggregating structured epithelial units to enable cell motility and morphogenesis occurring in the restricted events such as embryonic development and wound healing. For the retention of epithelial integrity in the adult organism, E-cadherin plays an important role for the formation of stable cell-cell adhesion. Loss of E-cadherin expression is a primal molecular event on the progression of tumour. 1 During the invasive process, tumour cells frequently undergo EMT, which comprises breakdown of E-cadherin-mediated interaction as an essential process. 2 Snail, a zinc-finger transcription factor, triggers EMT through direct repression of E-cadherin. 3,4 dEF-1, Twist and TGF-b also induce EMT in tumour cells and it accelerates malignancy of tumour. 5,6 In addition, maintenance of cell polarity is also fundamental for the identity of epithelial tissues. DNp63a, the predominant isoform of p63 which belongs to the p53 family, in basal keratinocytes plays a crucial role in the formation of stratified squamous epithelial structures by regulating asymmetric division. 7 We previously found that loss of p63 is accompanied by EMT in human squamous cell carcinoma (SCC) cells, and demonstrated that forced depletion of p63 leads to the acquisition of high-invasive capability independent of E-cadherin. 8 Significantly, the fact that re-exp...

Abstract. Receptor for hyaluronan-mediated motility (RHAMM) has previously been characterized as a cell surface receptor for hyaluronan and a microtubuleassociated intracellular hyaluronan binding protein. We examined the expression of RHAMM mRNA in 43 oral squamous cell carcinomas (SCCs) and 7 normal gingivae by real-time RT-PCR. The expression level of RHAMM mRNA was significantly higher in oral SCCs than normal gingivae (P=0.0047). Forty out of 43 oral SCCs showed expression of RHAMM splice variant (48 bp deletion). We immunohistochemically confirmed the protein expression of RHAMM in oral SCCs with higher levels of RHAMM mRNA. Patients with oral SCC who had high RHAMM expression had shorter survival rates than patients with low expression. However, it was not statistically significant. It has been reported that RHAMM interacts with spindle assembly factors such as microtubule-associated protein (TPX2). To investigate the expression of microtubuleassociated protein in oral SCCs, mRNA expression of TPX2 was also examined by real-time RT-PCR. The expression level of TPX2 mRNA was significantly higher in oral SCCs than normal gingivae (P=0.046). Furthermore, a significant correlation between the mRNA expression levels of TPX2 and RHAMM was recognized (P=0.011). The results indicate that there is a strong correlation between the mRNA expression levels of TPX2 and RHAMM in oral SCCs. Our observations suggest that the up-regulations of human RHAMM and TPX2 gene correlate with the malignant condition and might be linked to the increased or abnormal cell proliferation in human oral SCCs.

Th1 and Th2 cytokines such as interferon-gamma (IFN-gamma ) , tumor necrosis factor- alpha (TNF-alpha ), 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-gamma 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-alpha 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-gamma in RT7 cells, whereas IL-4 inhibited the increased levels by IFN-gamma and TNF-alpha in GT1 cells. Thus, IFN-gamma , TNF-alpha , and IL-4 appear cooperatively to regulate CXCR3-agonistic chemokines in oral keratinocytes and fibroblasts in T-cell-mediated oral inflammation sites.

Abstract. TPX2 is a microtubule-associated protein and is required for microtubule formation at kinetochores in mammalian cells. The purpose of this study was to clarify the expression of TPX2 mRNA and correlation between TPX2 and clinicopathological factors in salivary gland carcinomas. The expression of TPX2 mRNA was investigated in 20 human salivary gland carcinomas (8 mucoepidermoid carcinomas, 7 adenoid cystic carcinomas, 5 acinic cell carcinomas) and 6 normal submandibular glands using real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). The mean expression level of TPX2 mRNA was higher in mucoepidermoid carcinomas (0.53±0.51) than in normal submandibular glands (0.047±0.029); a significant association was found (Mann-Whitney U test, P=0.0067). The mean expression levels of TPX2 were also higher in acinic cell carcinomas (0.45±0.49) and adenoid cystic carcinomas (0.28±0.22) than in normal submandibular glands. Statistical correlations were found (Mann-Whitney U test, P=0.028 and P=0.003, respectively). Correlation between expression of TPX2 and receptor for hyaluronan-mediated motility (RHAMM) was also investigated in this study. A significant association was found between the mRNA expression levels of TPX and RHAMM (Pearson's correlation coefficient by rank test, P=0.020). These results indicate that human TPX2 mRNA is closely linked to increased or abnormal cell proliferation in malignant salivary gland tumors.