Down-regulated expression of SERPIN genes located on chromosome 18q21 in oral squamous cell carcinomas.

Serpins (serine protease inhibitors) are known as a diverse family of protease inhibitors; however, various other biological activities including tumor suppression, have been recently reported for these molecules. To clarify whether members of the serpin family are involved in OSCC (oral squamous cell carcinoma), global gene screening using microarray analysis was performed with OSCC-derived cell lines. A trend toward diminished expression was shown for some SERPIN genes located on 11q12-q13.1 and 18q21. mRNA expression of SERPIN genes at these chromosome regions was therefore analyzed using real-time quantitative RT-PCR (qRT-PCR) in 55 OSCC samples and matched normal tissue. Statistically significant decreases in expression were found for SERPINB12 (P=0.001), SERPINB13 (P=0.001), SERPINB4 (P=0.042), SERPINB3 (P<0.001), SERPINB11 (P<0.001), SERPINB7 (P=0.021) and SERPINB2 (P=0.018). All of these genes are located on 18q21, the known location of the serpin gene cluster. The results strongly suggest that this chromosome region plays a crucial role in OSCC. Some serpin members in the region might be involved in tumor suppression, or there might be unidentified tumor suppressor genes within or near the chromosome region.

Alteration of extracellular superoxide dismutase expression is associated with an aggressive phenotype of oral squamous-cell carcinoma.

Oxidative stress results in damage to cellular structures and has been linked to numerous diseases, including cancer. Extracellular superoxide dismutase (EC-SOD) is a principal enzymatic antioxidant in extracellular space. The purpose of this study was to determine whether the expression of EC-SOD protein is altered in the carcinogenetic process of oral squamous-cell carcinoma (OSCC). Immunohistochemical analysis was carried out in matched normal and tumour specimens collected from 58 OSCCs and 20 oral premalignant lesions (OPLs). Correlations between the EC-SOD expression levels and clinicopathological features of OSCC patients were evaluated by Fisher's exact test. Although EC-SOD protein was consistently expressed on the plasma membrane of cells in normal tissues, plasma membranous EC-SOD expression was lost in almost all the OSCC specimens examined (98%). Instead, positive EC-SOD expression was detected in the cytoplasmic compartments of cancerous cells in both OPLs (65%) and OSCCs (52%), together with a high incidence of lymph node metastasis (p=0.0397). These results suggest that the dysregulation of EC-SOD protein expression is a frequently occuring and early event in oral carcinogenesis, and that cytoplasmic EC-SOD may contribute to the increased aggressiveness of OSCC.

Characterization of intracellular superoxide dismutase alterations in premalignant and malignant lesions of the oral cavity: correlation with lymph node metastasis.

PURPOSE: The purpose of this study was to characterize changes in the expression of copper-zinc superoxide dismutase (Cu/Zn-SOD) and manganese SOD (Mn-SOD) in oral squamous-cell carcinoma (OSCC). METHODS: Real-time quantitative reverse transcriptase-polymerase chain reaction analysis of Cu/Zn-SOD and Mn-SOD mRNA expression was carried out in 50 pairs of OSCC tissue specimens and corresponding normal tissues. Mn-SOD protein expression was evaluated further in 65 OSCC tissue samples and 33 oral premalignant lesions (OPLs) using immunohistochemistry. RESULTS: Significant (P < 0.001) upregulation of Mn-SOD mRNA expression was observed in OSCC tissues compared with the normal tissue counterparts, whereas no significant difference was detected in Cu/Zn-SOD expression. Significant increases in Mn-SOD protein expression were seen in both OPLs (P < 0.001) and OSCC tissue (P < 0.001) together with a high incidence of lymph node metastasis (P = 0.04). CONCLUSIONS: Our findings suggested that Mn-SOD overexpression is a frequent and early event during oral carcinogenesis and could contribute to aggressive OSCC.

Down-regulation of plasma membranous Annexin A1 protein expression in premalignant and malignant lesions of the oral cavity: correlation with epithelial differentiation.

PURPOSE: To determine the potential involvement of ANXA1 in oral squamous-cell carcinoma (OSCC), we evaluated the ANXA1 protein expression in oral premalignant lesions (OPLs) and OSCCs and correlated the results with clinicopathologic variables. METHODS: Matched normal and tumour specimens of 44 primary OSCCs and 28 OPLs were analyzed for ANXA1 subcellular localization and protein expression level by immunohistochemistry (IHC). Correlations between ANXA1-IHC staining scores of OSCCs and clinicopathologic features were evaluated by Fisher's exact test. RESULTS: Markedly down-regulation of ANXA1 protein expression was identified on the plasma membrane of epithelial cells in OSCCs (P < 0.001) and OPLs (P = 0.001) compared with normal counterparts. Moreover, loss of plasma membranous ANXA1 expression was significantly correlated with the poorly differentiated status of OSCC cells (P = 0.012). CONCLUSIONS: Our findings suggest that loss of ANXA1 is frequent and early event during oral carcinogenesis and that ANXA1 could contribute to maintaining epithelial differentiation in OSCC.

Overexpression and altered subcellular localization of autophagy-related 16-like 1 in human oral squamous-cell carcinoma: correlation with lymphovascular invasion and lymph-node metastasis.

Autophagy is a dynamic process of subcellular degradation, which has recently sparked great interest because it is involved in various developmental processes and various diseases including cancer. Autophagy-related 16-like 1 is a component of a large protein complex essential for autophagosome formation. We previously applied proteomic methods to characterize differentially expressed proteins in oral squamous cell carcinoma cells and detected significantly high expression levels of autophagy-related 16-like 1 in oral squamous cell carcinoma-derived cell lines compared to human normal oral keratinocytes. In the current study, to further determine the potential involvement of autophagy-related 16-like 1 in oral squamous cell carcinoma, we evaluated the state of autophagy-related 16-like 1 protein expression in human oral premalignant lesions and primary oral squamous cell carcinomas, and correlated the results with clinicopathologic variables. Autophagy-related 16-like 1 immunoreaction was predominant in a variety of subcellular components of oral squamous cell carcinoma tissues, including the cytoplasm and plasma membrane of malignant cells (45% and 39%, respectively) and peritumoral and intratumoral stroma (52%), whereas all of the components in normal tissues had no or faint autophagy-related 16-like 1 expression. In addition, high stromal expression of autophagy-related 16-like 1 was associated significantly with lymphovascular invasion of tumor cells (P = .037) and positive lymph node status (P = .015). Furthermore, cytoplasmic and plasma membranous autophagy-related 16-like 1 were also expressed in abundance in the oral premalignant lesion cells (74% and 32%, respectively). Our finding suggests that dysregulation of autophagy-related 16-like 1 protein expression is a frequent and early event during oral carcinogenesis and could affect the malignant behavior of oral squamous cell carcinoma cells.

Hyaluronan-mediated motility: a target in oral squamous cell carcinoma.

To characterize cancer-related gene expression changes in oral squamous cell carcinomas (OSCCs), we compared the gene expression profiles in OSCC-derived cell lines with human normal oral keratinocytes (HNOKs). Microarray analysis identified 166 genes that were up-regulated in OSCC-derived cell lines. Gene ontology analysis showed that cancer-related function had the highest significance. Among the genes mapped to the cancer-related network with the highest significance, the receptor for hyaluronan-mediated motility (RHAMM) was evaluated further for mRNA and protein expression in the OSCC cell lines, primary OSCCs. Overexpression of RHAMM protein was observed in all cell lines compared to HNOKs. Immunohistochemical analysis showed highly expressed RHAMM in primary OSCCs, whereas most corresponding normal tissues had no or significant down-regulation of protein immunoreactivity. Real-time quantitative reverse transcriptase-polymerase chain reaction data agreed with the protein expression. Moreover, the RHAMM expression status was correlated with the TNM stage (P<0.001). The results suggested that RHAMM expression may be correlated with tumor aggressiveness and offer clues to the development of new treatments for human OSCCs.

Clinical significance of gelsolin-like actin-capping protein expression in oral carcinogenesis: an immunohistochemical study of premalignant and malignant lesions of the oral cavity.

BACKGROUND: Gelsolin-like actin-capping protein (CapG) is a ubiquitous gelsolin-family actin-modulating protein involved in cell signalling, receptor-mediated membrane ruffling, phagocytosis, and motility. CapG has generated great interest due to its oncogenic function in the control of cell migration or invasion in a variety of cancer cells. We previously applied proteomic methods to characterize differentially expressed proteins in oral squamous-cell carcinoma (OSCC) cells and detected significantly high expression levels of CapG in OSCC-derived cell lines compared to human normal oral keratinocytes. In the current study, to further determine the potential involvement of CapG in OSCC, we evaluated the status of CapG protein and mRNA expression in human oral premalignant lesions (OPLs) and primary OSCCs and correlated the results with clinicopathologic variables. METHODS: Matched normal and tumour tissue sections of 79 human primary OSCCs and 28 OPLs were analyzed for CapG expression by immunohistochemistry (IHC). Correlations between CapG-immunohistochemical staining scores of OSCCs and clinicopathologic features were evaluated by Fisher's exact test. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was used to estimate CapG expression at the mRNA level. RESULTS: In IHC, substantial up-regulation of CapG protein was observed in primary OSCCs (52%) and OPLs (64%), whereas corresponding normal tissues showed consistently weak or absent immunoreactivity of CapG. qRT-PCR data were consistent with the protein expression status. Moreover, CapG expression was correlated with the TNM stage grading of OSCCs. CONCLUSION: Our finding of frequent dysregulated expression of CapG in premalignant and malignant lesions together with an association with an advanced clinical disease stage suggests that CapG could contribute to cancer development and progression and that CapG may have potential as a biomarker and a therapeutic target for OSCC.

Genes and molecular pathways related to radioresistance of oral squamous cell carcinoma cells.

To identify genes associated with radioresistant oral squamous cell carcinoma (OSCC), we compared gene expression signatures between OSCC cell lines exhibiting radioresistance and cells with radiosensitivity after X-ray irradiation in a dose-dependent manner using Affymetrix GeneChip analysis with Human Genome-U133 plus 2.0 GeneChip. The microarray data identified 167 genes that were significantly overexpressed in radioresistant cells after X-ray irradiation. Among the genes identified, 40 were mapped to 3 highly significant genetic networks identified by the Ingenuity Pathway Analysis tool. Gene ontology analysis showed that cancer-related function had the highest significance. The 40 genes included 25 cancer-related genes that formed 1 network and were categorized by function into growth and proliferation, apoptosis, and adhesion. Furthermore, real-time quantitative reverse transcriptase-polymerase chain reaction showed that the mRNA expression levels of the 25 genes were higher in radioresistant cells than in radiosensitive cells in a dose-dependent manner and in a time-dependent manner. Our results suggest that the identified genes help to elucidate the molecular mechanisms of the radioresistance of OSCC and could be radiotherapeutic molecular markers for choosing the appropriate radiotherapy for this disease.