齋藤 謙悟

Treatment protocols for malignant tumors in the oral cavity differ greatly based on the presence of cervical lymph node metastasis. We applied gene expression profiling to the pathological lymph node status and used a training-test approach to evaluate the reliability of cDNA microarray-based classifications of 15 matched resected primary oral squamous cell carcinomas (OSCCs) and corresponding normal oral tissues. The clustering of all the microarray data was separated into two groups based on metastatic node positivity and node negativity. Furthermore, a 20-gene signature was identified that differentiated the testing set (n=8) with high classification accuracy (88%). Our findings support the hypothesis that the lymph node metastasis status can be predicted using the gene expression patterns of the primary OSCC, and may be a powerful tool in identifying patients at high risk of lymph node metastasis.

Functional proteomics is a useful method to explore changes in protein expression in human diseases, including carcinomas. To identify tumor-associated proteins as biomarkers or molecular targets of human oral squamous cell carcinomas (OSCCs), we performed two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Comparison of the protein expression profiles of OSCC cell lines and normal oral keratinocytes identified six proteins with markedly different expression levels. Of the six proteins, we found a 1D-myo-inositol 1,4,5-trisphosphate 3-kinase A (ITPKA) protein that was down-regulated in OSCC cell lines. ITPKA phosphorylates inositol 1,4,5-trisphosphate, which regulates the calcium (Ca2+) level within the cell by releasing Ca2+ from intracellular stores, and is responsible for regulating the levels of a large number of inositol polyphosphates that are important in cellular signaling. Western blots revealed dramatically down-regulated ITPKA expression in all OSCC cell lines examined. Real-time quantitative reverse transcriptase-polymerase chain reaction showed down-regulated ITPKA mRNA expression in nine of 12 (75%) OSCC cell lines. Immunohistochemistry analysis showed that 40 of 100 OSCC clinical samples had a significant decrease in ITPKA. Poorly differentiated tumors showed significantly lower immunoreactivity of the protein compared to well- and moderately-differentiated tumors. These data suggest that ITPKA may be related to carcinogenesis by the modulation of inositol polyphosphates and Ca2+ homeostasis and that ITPKA may be a potential novel molecular target, biomarker, parameter, or all of these of cellular differentiation and of intracellular Ca2+ homeostatic characteristics in clinical medicine.

Stathmin is an intracellular phosphoprotein that is overexpressed in a number of human malignancies. Our previous study using proteomic profiling showed that significant upregulation of stathmin occurs in oral squamous-cell carcinoma ( OSCC)-derived cell lines. In the current study, to determine the potential involvement of stathmin in OSCC, we evaluated the state of stathmin protein and mRNA expression in OSCC-derived cell lines and human primary OSCCs. A significant increase in stathmin expression was observed in all OSCC-derived cell lines examined compared to human normal oral keratinocytes. In immunohistochemistry, 65% of the OSCCs were positive for stathmin, and no immunoreaction was observed in corresponding normal tissues. Real-time quantitative reverse transcriptase-polymerase chain reaction data were consistent with the protein expression status. Moreover, stathmin expression status was correlated with the TNM stage grading. Furthermore, we found a statistical correlation between the protein expression status and disease-free survival ( P = 0.029). These results suggest that expression of stathmin could contribute to cancer progression/prognosis, and that stathmin may have potential as a biomarker and a therapeutic target for OSCC.

Adenoid cystic carcinoma (ACC) is one of the most common malignant tumors of the salivary glands. It tends to grow slowly but is associated with a poor prognosis compared to other malignant salivary gland tumors. To identify specific markers of ACC, we examined protein expression profiling in ACC xenograft and normal salivary glands (NSG) using fluorescent 2-dimensional differential in-gel electrophoresis (2-D-DIGE), an emerging technique for comparative proteomics, that improves the reproducibility and reliability of differential protein expression analysis between the samples. To identify the proteins, matrix-assisted laser desorption/ionization time-of-flight peptide mass fingerprinting was carried out. Using these strategies, we detected 4 upregulated proteins and 5 downregulated proteins in ACC xenograft. Maspin and stathmin were selected for further analyses. Western blotting and immunohistochemical staining showed a higher expression of these proteins in ACC xenograft and clinical ACC tissue compared to NSG. Furthermore, Expression of these proteins was correlated with the histologic grading of ACC (n = 10). Therefore, our data indicate that maspin and stathmin may be not only useful biomarkers of ACC but also markers of biologic behavior in this tumor. (c) 2005 Wiley-Liss, Inc.

The plasma membrane Ca2+ ATPase (PMCA) is an essential regulator of free intracellular calcium. Recent studies have reported aberrant expression of the PMCA1 gene, a member of the PMCA family, in several cancer cell types. To elucidate the contribution of PMCA1 to oral carcinogenesis, we analyzed genetic and epigenetic changes and mRNA and protein expression in primary oral squamous cell carcinomas (OSCCs), oral premalignant lesions (OPLs), and OSCC-derived cell lines. The PMCA1 gene was epigenetically inactivated, but not mutated in the eight OSCC-derived cell lines tested. In clinical samples, frequent down-regulation of PMCA1 protein expression was found not only in primary OSCCs (43%), but also in OPLs (40%). Real-time quantitative reverse transcriptase-polymerase chain reaction data were consistent with the protein expression status. These results suggest that inactivation of the PMCA1 gene is a frequent and early event during oral carcinogenesis, and gene expression may be regulated by an epigenetic mechanism.