DeltaNp63alpha-dependent expression of Id-3 distinctively suppresses the invasiveness of human squamous cell carcinoma.

p63 is a member of the p53 family and DeltaNp63alpha 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 DeltaNp63alpha diminished their invasiveness (Higashikawa K, Yoneda S, Tobiume K, Taki M, Shigeishi H, Kamata N. Snail-induced down-regulation of DeltaNp63alpha acquires invasive phenotype of human squamous cell carcinoma. Cancer Res 2007;67:9207-13). In this study, we found that DeltaNp63alpha 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 DeltaNp63alpha-Id-3 axis incident to Snail-mediated EMT for the MMP-2-dependent invasiveness in SCC cells.

Snail-induced down-regulation of DeltaNp63alpha 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.

Optical surgical navigation-assisted removal of a foreign body using a splint to simplify the registration process: a case report.

BACKGROUND: Oral and maxillofacial surgeons often encounter foreign objects within the human body. Despite the visual identification of foreign objects via imaging techniques, the accurate determination of their position in the maxillofacial area can be challenging. The clinical application of a navigation system can solve this issue. This system provides a useful guide for a safer and more accurate surgical technique by accurately determining the location of the lesion in real time during the surgery. However, complications with regard to registration may be encountered. We describe a navigation system that simplifies registration using a dental splint with embedded reference points for foreign body removal in the maxilla. CASE PRESENTATION: A 78-year-old Japanese woman was referred with the chief complaint of pain in the left upper molar region. We found the symptoms to be associated with a foreign body in the maxilla and decided to remove it. A minimally invasive treatment procedure was desirable. However, the lesion was in contact with the maxillary sinus, and it was difficult to pinpoint its position because of the absence of an anatomical landmark. Therefore, we decided to use a navigation system. In order to simplify registration, a dental splint with embedded reference points was created. The registration could be reliably performed before surgery using an optical navigation system that facilitates the process, using splints with embedded reference points. Following preoperative registration, the splint with the reference frame was placed in the patient's mouth, and the accuracy of the navigation was confirmed. The position with respect to the maxillary sinus was precisely identified followed by the removal of the surrounding bone and excision of the lesion. Therefore, the surgery could be accurately performed without perforating the maxillary sinus. In addition, owing to preoperative registration, the operative time could be shortened. After the surgical procedure, the patient's symptoms disappeared. CONCLUSIONS: The procedure was performed in a precise, minimally invasive manner. Furthermore, the operative time was reduced by the simplified registration process, wherein a splint was embedded with reference points. This technique may prove useful for performing maxillofacial surgical procedures.

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.

Up-regulation of stromal cell-derived factor-1alpha and its receptor CXCR4 expression accompanied with epithelial-mesenchymal transition in human oral squamous cell carcinoma.

Stromal cell-derived factor 1alpha (SDF-1alpha) and its receptor CXCR4 have been implicated in the tumorigenesis, proliferation, and lymph node metastasis of cancer. Here, we report that highly invasive squamous cell carcinoma (SCC) cells with a spindle cell morphology show a strong expression of both SDF-1alpha and CXCR4. CXCR4 expression and cell migratory activity were further up-regulated by treatment with SDF-1alpha or TGF-beta1 in these cells. When epithelial-mesenchymal transition (EMT) was induced by Snail over-expression in SCC cells with an epithelial phenotype, an increased expression of SDF-1alpha was observed. Furthermore, SDF-1alpha and TGF-beta1 up-regulated the expression of CXCR4 and cell migratory activity in these cells. These results indicate that SDF-1alpha and CXCR4 expressions are possible markers of highly-invasive SCC and regulated by EMT.

Increased expression of CENP-H gene in human oral squamous cell carcinomas harboring high-proliferative activity.

We examined the expression of Centromere protein H (CENP-H) mRNA in 38 oral squamous cell carcinomas (SCCs), 2 epithelial dysplasias and 5 normal gingivae using the real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). The mean expression level of CENP-H mRNA was higher in oral SCCs (0.11+/-0.08) than epithelial dysplasias (0.03+/-0.01) and normal gingivae (0.027+/-0.01). The expression level of CENP-H mRNA was significantly higher in oral SCCs than normal gingivae (Mann-Whitney U test, P=0.005). We also found a significant association between the level of CENP-H mRNA expression and clinical stage in oral SCCs (Mann-Whitney U test, P=0.04). We next studied the expression of CENP-H in 17 oral SCCs immunohistochemically. A significant correlation between the expression levels of CENP-H protein and the Ki-67 labeling index was found (Mann-Whitney U test, P=0.005). These results indicate that human CENP-H is closely linked to the increased or abnormal cell proliferation in malignant conditions.

Correlation of human Bub1 expression with tumor-proliferating activity in salivary gland tumors.

Human Bub1 plays an important role at the spindle assembly check-point to prevent cell cycle progression following spindle damage. We examined the expression of Bub1 mRNA and protein in 21 human salivary gland tumors (7 pleomorphic adenomas, 2 warthin tumors, 5 mucoepidermoid carcinomas, 3 adenoid cystic carcinomas and 4 acinic cell carcinomas) and 3 normal submandibular glands using real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) or western blotting. The mean expression levels of Bub1 mRNA and protein were higher in malignant tumors (0.12+/-0.028/1.75+/-0.53) than normal submandibular glands (0.042+/-0.014/0.19+/-0.044) and benign tumors (0.058+/-0.01/0.97+/-0.44). We found a significant association between the level of Bub1 mRNA/protein expression and clinical stage in malignant tumors (Mann-Whitney U test, p=0.019/p=0.016). We analyzed its relation with the proliferative activity monitored by the Ki-67 labeling index by immunohistochemistry as well as the expression of proliferating cell nuclear antigen (PCNA) by Western blotting. A significant correlation was found between Bub1 mRNA/protein expression and the Ki-67 labeling index in salivary gland tumors (Spearman's correlation coefficient by rank test, p=0.026/p=0.002). These results indicate that increased expression of the human Bub1 gene is closely linked to abnormal cell proliferation in malignant conditions.

Correlation of CENP-F gene expression with tumor-proliferating activity in human salivary gland tumors.

We examined the expression of Centromere protein F (CENP-F) mRNA in 26 human salivary gland tumors (seven pleomorphic adenomas, three Warthin tumors, seven mucoepidermoid carcinomas, four adenoid cystic carcinomas, four acinic cell carcinomas and one malignant myoepithelioma) and four normal submandibular glands using the real time quantitative reverse transcription-polymerase chain reaction (RT-PCR). The mean expression level of CENP-F mRNA was higher in malignant tumors (1.05+/-0.32) than normal submandibular glands (0.11+/-0.05) and benign tumors (0.46+/-0.16). We found a significant association between the level of CENP-F mRNA expression and clinical stage in 16 malignant tumors (Mann-Whitney U test, p=0.027). We also found a significant correlation between the Ki-67 labeling index and CENP-F expression in malignant tumors (Spearmans correlation coefficient by rank test, p=0.029). These results indicate that human CENP-F mRNA is closely linked to the increased or abnormal cell proliferation in malignant conditions.