Satoru Shintani

Inactivation of the p14ARF, p15INK4B and p16INK4A genes is a frequent event in human oral squamous cell carcinomas

The p14(ARF), p15(INK4B) and p16(INK4A) genes were localized to 9p21, where genetic alterations have been reported frequently in various human tumors. We performed a molecular analysis of the mechanism of inactivation in cell lines and 32 oral squamous cell carcinoma (OSCC), using deletion screening, PCR-SSCP, methylation-specific-PCR and cycle sequencing. We detected homozygous deletion of p14(ARF)-1Ebeta in 9 (26.5%), of p15(INK4B) in one (3.1%), and of p16(INK4A) in 22 (56.3%) tumor samples. Three mutations were detected in the p16(INK4A) genes. We detected aberrant methylation of the p14(ARF) genes in 14 (43.8%), of the p15(INK4B) gene in 9 (28.1%), and of the p16(INK4A) gene in 16 (50.0%) tumor samples. Altogether, 87.5% of the samples harbored at least one of the alterations in the p14(ARF), p15(INK4B), and p16(INK4A) genes, indicating that the frequent inactivation of these genes may be an important mechanism during OSCC development.

Anti-Epidermal Growth Factor Receptor Monoclonal Antibody 225 Upregulates p27KIP1 and p15INK4B and Induces G1 Arrest in Oral Squamous Carcinoma Cell Lines

Epidermal growth factor receptor (EGFR) regulates the growth and progression of human oral squamous cell carcinoma (SCC). Recently, the link between EGFR signaling and the cell cycle has been identified. Some reports have described that EGFR-blocking monoclonal antibody 225 (mAb225) induced G1 arrest and inhibited the growth of various cancer cells. The purpose of this study was to evaluate the effect of mAb225 on human oral SCC cell lines. Exposure to mAb225 in culture inhibited the growth of oral SCC cell lines in an EGFR number-independent manner, with the percent inhibition ranging from 13.8 to 76.6%. Flow-cytometric analysis demonstrated that treatment with mAb225 induced cell accumulation in G1 phase, accompanied by a decrease in the percentage of cells in the S phase. Apoptosis was not seen in this study. G1 arrest was accompanied by a decrease in CDK2-, CDK4-, and CDK6-associated histone H1 kinase activities, and an increase in the expression levels of cell cycle inhibitors p27KIP1 and p15INK4B. These results suggested that the antiproliferative effect of EGFR blockade by mAb225 in oral SCC may be mediated by p27KIP1 and p15INK4B.

High frequency of homozygous deletion and methylation of p16INK4A gene in oral squamous cell carcinomas

p16(INK4A) inactivation was analyzed in ten squamous cell carcinoma (SCC) cell lines and 32 primary SCCs, using the polymerase chain reaction (PCR), PCR-single-strand conformation polymorphism, methylation-specific PCR, and cycle sequencing. In the study of cell lines, we detected three deletions in exon 1alpha and exon 2, and detected two methylations. Among tumor samples, we detected the homozygous deletions (HDs) of 43.8% in exon 1alpha 34.4% in exon 2, and methylation was found in 50.0%. The lack of p16(INK4A) with immunohistochemistry was detected in 71.9% and matched the alteration of p16(INK4A) gene. These results suggest that p16(INK4A) inactivation is predominantly caused by HD and methylation, and immunohistochemical evaluation of p16(INK4A) is a useful method.

Alterations of Rb, p16INK4A and cyclin D1 in the tumorigenesis of oral squamous cell carcinomas

Immunohistochemical analysis of Rb, p16INK4A and cyclin D1 expression was performed on 78 oral squamous cell carcinoma (SCC), 46 leukoplakia, and 20 normal mucosa. Rb and p16INK4A expression were observed in all normal mucosa and most of leukoplakia. Lack of Rb and p16INK4A was observed in 56.4 and 67.9% of SCC, respectively. The overexpression of cyclin D1 was not observed in normal mucosa and was observed in 35.9% of SCC. A strong reciprocal relationship between Rb and p16INK4A expression was observed in oral SCC, and all these SCC cases have at least one of the alterations in the Rb pathway.

Prognostic significance of ERRB3 overexpression in oral squamous cell carcinoma

Immunohistochemical analysis of erbB3, as the third member of epidermal growth factor receptor gene family, was performed on 41 cases of oral squamous cell carcinoma, correlating the staining pattern with clinical outcome. High expression of erbB3 protein (ERBB3) was significantly associated with lymph node metastasis (P < 0.05), survival rate (P < 0.05) and mode of invasion (P < 0.01) in this series. These results demonstrated that ERBB3 expression may be helpful in identifying those oral squamous cell carcinomas with higher malignant potential.

Overexpression of CDK2 is a prognostic indicator of oral cancer progression.

Cyclins and cyclin‐dependent kinases (CDKs) play key roles in cell cycle regulation, a process of which dysregulation can lead to uncontrolled cell growth and hence to cancer. We have already reported the alteration of CDK4 and cyclin Dl expression in oral cancer. In this study, we examined by immunohistochemistry the expression of CDK2, and cyclins A and E in 20 normal oral mucosa, 42 dysplastic epithelia, and 103 oral squamous cell carcinomas (SCCs). The expressions of CDK2, and cyclins A and E were not detected in the normal epithelium and significantly altered from epithelial dysplasia to SCC. While there were no significant correlations between the expression of cyclins A, E and the patients survival, CDK2 expression was significantly correlated with lymph node involvement (P=0.025), tumor differentiation (P=0.032), mode of tumor invasion (P=0.017), and shorter survival period (P=0.0173). These results suggest that the elevated expression of CDK2 is a critical factor in oral cancer progression and can be used as a negative predictive marker of the patients prognosis.

p53 and MDM2 Expression in Oral Squamous Cell Carcinoma

The p53 tumor suppressor gene is the most commonly mutated gene in human cancer and is a frequent abnormality in oral squamous cell carcinoma and its precancerous lesions. MDM2 (murine double minute-2), a new proto-oncogene, may be associated with p53 gene products and may negatively affect the transcriptional activating function of p53. The purpose of this study was to investigate the incidence of MDM2 and its relationship to the expression of p53 in oral squamous cell carcinoma and precancerous lesions. Overexpression of p53 and MDM2 proteins was detected in 52 and 40% of oral squamous cell carcinomas, respectively. p53 gene mutation, absent in normal oral epithelium was observed in 31% of the carcinoma cases. Our finding suggested that MDM2 protein may be an alternative mechanism causing p53 protein dysfunction in oral squamous cell carcinoma.

Extracellular matrices expression in invasion area of adenoid cystic carcinoma of salivary glands

Adenoid cystic carcinoma (ACC) is a salivary malignant tumor with poor long-term prognosis, that is known to have predilection for invasion of the adjacent stroma and neural tissues. This carcinoma has shown a high incidence of recurrence and distal metastasis. Invasive carcinomas have been associated with the distributions of extracellular matrices (ECM). Cell proliferation as a marker of tumor growth has been related to poor prognosis in oral carcinomas. Immunohistochemical analysis of 15 cases of ACC was done using antibodies to laminin, type IV collagen, fibronectin, tenascin and anti-proliferating nuclear antigen (PCNA). Laminin and type IV collagen were totally or partially absent in the ACC invasive areas. Tenascin was expressed in the stroma and cytoplasm and was associated with tumor cell proliferation. It can be concluded that basement membrane represents a barrier that is lost during cell invasion and tenascin may be involved in the detachment of cancer cells, increasing the invasive potential of ACC.

Association of Preoperative Radiation Effect with Tumor Angiogenesis and Vascular Endothelial Growth Factor in Oral Squamous Cell Carcinoma

This study examined the relationship between tumor angiogenesis and the radiation‐induced response, evaluated based on pathological changes, in oral squamous cell carcinoma patients treated with preoperative radiation therapy. Forty‐one cases of squamous cell carcinoma treated with preoperative radiation therapy were investigated. Tumor angiogenesis was assessed by scoring the intratumor microvessel density (IMVD). Expression of vascular endothelial growth factor (VEGF) was also evaluated before and after preoperative radiotherapy. There was no correlation between IMVD in the specimens before therapy and the pathological response to radiation therapy. However, radiation therapy decreased IMVD in the specimens after therapy. A significant association was observed between VEGF expression and resistance to radiation therapy: only 4 of the 21 patients whose tumors exhibited a high level (2+ or 3+) of VEGF staining experienced a major (3+ or 4+) pathological response to radiation therapy. Furthermore, an increasing level of VEGF expression after radiation therapy was observed in non‐effective (0 to 2+) response cases. These results suggest that VEGF expression and the induction of this protein are related to radiosensitivity and could be used to predict the effects of preoperative radiation therapy on oral squamous cell carcinoma.

Apoptosis and p53 are associated with effect of preoperative radiation in oral squamous cell carcinomas.

This study was carried out to elucidate whether apoptosis and p53 can be used to stratify oral cancer patients into groups with a favorable or unfavorable response to preoperative radiation therapy. Thirty-two patients were evaluated. The apoptosis index was 1.7+/-0. 9% in the ineffective cases, and it was significantly lower than effective cases (3.2+/-1.2%). While 14 of 16 effective cases (86.7%) did not express p53, 13 of 16 ineffective cases (81.3%) overexpressed p53. These results suggest that mutated p53 in tumors is associated with a poor response to radiation which may be related to evasion of apoptosis in oral cancer.