Hidetaka Yokoe

Overexpression of stathmin in oral squamous-cell carcinoma: correlation with tumour progression and poor prognosis

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.

Sarcoendoplasmic reticulum Ca2+ atpase type 2 downregulated in human oral squamous cell carcinoma

Mice with a heterozygous deletion of the Atp2a2 gene (Atp2a2+/−) encoding SERCA2 spontaneously develop SCCs of the skin and upper digestive tract, including the oral cavity. To elucidate the contribution of ATP2A2 to human oral carcinogenesis, we analyzed genetic and epigenetic changes as well as mRNA and protein expression in primary OSCCs and OPLs. With the exception of one OSCC‐derived cell line showing a 12 bp deletion of ATP2A2, we found no mutations in the coding sequence of the gene in primary OSCCs (n = 52), OPLs (n = 32) and cell lines (n = 8). In immunohistochemistry, however, high frequencies of ATP2A2 downregulation were evident not only in primary OSCCs (42%, 42/100) but also in OPLs (31%, 10/32). Real‐time quantitative RT‐PCR data were consistent with the protein expression status. Aberrant DNA methylation within ATP2A2 also was detected in 9 of 30 ATP2A2‐downregulated OSCCs. Moreover, restoration or elevated expression of the ATP2A2 protein was induced in most of the cell lines showing ATP2A2 methylation after treatment with 5‐aza‐2′‐dC, a DNA demethylating agent. These results suggest that inactivation of the ATP2A2 gene is a frequent and early event during oral carcinogenesis and that loss of expression may be regulated partly by an epigenetic mechanism.

Aberrant expression of RAB1A in human tongue cancer

This study was designed to identify specific gene expression changes in tongue squamous cell carcinomas (TSCCs) compared with normal tissues using in-house cDNA microarray that comprised of 2304 full-length cDNAs from a cDNA library prepared from normal oral tissues, primary oral cancers, and oral cancer cell lines. The genes identified by our microarray system were further analysed at the mRNA or protein expression level in a series of clinical samples by real-time quantitative reverse transcriptase–polymerase chain reaction (qRT–PCR) analysis and imuunohositochemistry. The microarray analysis identified a total of 16 genes that were significantly upregulated in common among four TSCC specimens. Consistent with the results of the microarray, increased mRNA levels of selected genes with known molecular functions were found in the four TSCCs. Among genes identified, Rab1a, a member of the Ras oncogene family, was further analysed for its protein expression in 54 TSCCs and 13 premalignant lesions. We found a high prevalence of Rab1A-overexpression not only in TSCCs (98%) but also in premalignant lesions (93%). Thus, our results suggest that rapid characterisation of the target gene(s) for TSCCs can be accomplished using our in-house cDNA microarray analysis combined with the qRT–PCR and immunohistochemistry, and that the Rab1A is a potential biomarker of tongue carcinogenesis.

Expression of an Inhibitor of Apoptosis, Survivin, in Oral Carcinogenesis

A novel inhibitor of apoptosis, survivin, plays a role in oncogenesis. To determine the potential involvement of survivin in oral carcinogenesis, we investigated the distribution of survivin protein expression in oral squamous cell carcinomas (OSCCs) and oral pre-malignant lesions. The mRNA expression level and methylation status of the gene also were evaluated in OSCCs and OSCC-derived cell lines. In immunohistochemistry, 58% of tumors and 37% of pre-malignant lesions examined were positive for survivin, while no immunoreaction was observed in corresponding normal tissues. The reverse-transcription/polymerase chain-reaction revealed similar changes in survivin gene expression levels. Furthermore, of the 9 normal oral tissues with no survivin gene expression, 4 showed methylation of the gene, while no methylation was detected in the corresponding tumorous tissues. The results suggest that survivin plays an important role during oral carcinogenesis, and that the gene expression may be regulated by an epigenetic mechanism.

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.

Ubiquitous mitochondrial creatine kinase downregulated in oral squamous cell carcinoma

In this study, we performed two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionisation time of fly mass spectrometry to identify the protein(s) associated with the development of oral squamous cell carcinomas (OSCCs) by comparing patterns of OSCC-derived cell lines with normal oral keratinocytes (NOKs), and found that downregulation of ubiquitous mitochondrial creatine kinase (CKMT1) could be a good candidate. Decreased levels of CKMT1 mRNA and protein were detected in all OSCC-derived cell lines examined (n=9) when compared to those in primary normal oral keratinocytes. Although no sequence variation in the coding region of the CKMT1 gene with the exception of a nonsense mutation in exon 8 was identified in these cell lines, we found a frequent hypermethylation in the CpG island region. CKMT1 expression was restored by experimental demethylation. In addition, when we transfected CKMT1 into the cell lines, they showed an apoptotic phenotype but no invasiveness. In clinical samples, high frequencies of CKMT1 downregulation were detected by immunohistochemistry (19 of 52 (37%)) and quantitative real-time RT–PCR (21 of 50 (42%)). Furthermore, the CKMT1 expression status was significantly correlated with tumour differentiation (P<0.0001). These results suggest that the CKMT1 gene is frequently inactivated during oral carcinogenesis and that an epigenetic mechanism may regulate loss of expression, which may lead to block apoptosis.

Relationship between the number of remaining teeth and physical activity in community-dwelling elderly.

The relationship between oral health and general health in the elderly has been much studied. However, further studies focussing on the influence of oral health on the quality of life (QOL) among the elderly are required. The goal of this study was to evaluate the relationship between oral health and physical or cultural activities. Subjects were 101 community-dwelling elderly persons who were functionally independent (mean age 70.3). Oral health status was evaluated according to the number of remaining teeth and the number of functional teeth. Physical and cultural activities were evaluated from self-reported information. The relationship between oral health and physical or cultural activities was examined by logistic regression analysis. About 60% of subjects took part in cultural activities, and less than half actively exercised (leisure sports, 33.6%; travel, 42.6%). Persons with 20 or more remaining teeth were more active in leisure sports (Odds ratio (OR)=4.86, 95% confidence interval (CI)=1.34, 17.38) and travel (OR=5.42, 95% CI=1.63, 18.08) than those with fewer than 20 remaining teeth. These results suggest that the number of remaining teeth is associated with physical activity in elderly persons.

Gingival squamous cell carcinoma in a patient with chronic graft-versus-host disease

This article describes a gingival squamous cell carcinoma that developed in a 21-year-old woman who received a bone marrow transplant at the age of 16 from her human leukocyte antigen-identical sister as treatment for severe aplastic anemia. Thirty days after transplantation, she presented with cutaneous erythema as a result of acute graft-versus-host disease, and this subsequently evolved into chronic graft-versus-host disease. A lichenoid white plaque of the gingiva developed shortly thereafter, and it began to increase in size rapidly 4 years posttransplantation. Biopsy indicated squamous cell carcinoma arising in this region, apparently associated with chronic graft-versus-host disease. Few reports have described a secondary solid malignancy involving the oral cavity of young adults after bone marrow transplantation.

Localization of a tumour-suppressor gene associated with human oral cancer on 7q31.1.

To search for the existence of a tumour‐suppressor gene (TSG) associated with oral squamous cell carcinoma (SCC), PCR analysis of microsatellite polymorphisms corresponding to 14 loci which map to chromosome 7q21.3‐qter was performed to screen 35 patients with oral SCC for loss of heterozygosity (LOH). LOH was observed in at least one of the loci in 19 of 34 (55.9%) informative cases. Among the loci tested, frequent LOH was restricted at D7S522 on chromosome 7q31.1, which was measured within 1 cM. Furthermore, we detected microsatellite instability (MI) in 11 of 35 (31.4%) cases tested. Our observations indicate that alterations of chromosome 7q are associated with oral SCC tumorigenesis and that 7q31.1 might harbour at least one putative TSG. Int. J. Cancer 75:671–674, 1998.© 1998 Wiley‐Liss, Inc.

Monitoring of circulating tumour-associated DNA as a prognostic tool for oral squamous cell carcinoma

Frequent allelic imbalances (AIs) including loss of heterozygosity and microsatellite instability on a specific chromosomal region have been identified in a variety of human malignancies. The objective of our study was to assess the possibility of prognostication and monitoring of oral squamous cell carcinoma (SCC) by microsatellite blood assay. DNA from normal and tumorous tissues and serum DNA obtained at three time points (preoperatively, postoperatively, and 4 weeks postoperatively) from 64 patients with oral SCC was examined at nine microsatellite loci. In all, 38 (59%) DNA samples from tumorous tissues and 52% from serum showed AIs in at least one locus. Patterns of AIs in the serum DNA were matched to those detected in tumour DNA. Of them, AIs were frequently detected preoperatively (44%, 28 of 64), and postoperatively (20%, 13 of 64). Moreover, among 12 cases with AIs during the postoperative period, six had no evidence of an AI 4 weeks postoperatively, and they had no recurrence and were disease free. In contrast, six patients with AI-positive DNA 4 weeks postoperatively have died with distant metastasis within 44 weeks. Thus, our results suggest that the assessment of microsatellite status in the serum DNA could be a useful predictive tool to monitor disease prognosis.