Masashi Shiiba

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.

Allelic loss of chromosome 13q14.3 in human oral cancer: Correlation with lymph node metastasis

To evaluate the role of chromosome 13 deletions in oral squamous cell carcinoma (SCC) progression and to define the precise localization of putative tumor suppressor genes, we studied tumors from 34 unrelated patients with oral SCC by the polymerase chain reaction (PCR)‐loss of heterozygosity (LOH) assay, using 18 different polymorphic loci. Chromosome 13q allelic losses (LOH) were observed in 67.6% at 1 or more loci. These results enabled the identification of a putative minimal region of deletion mapped at 13q14.3. The commonly deleted region is located close, but telomeric to the RB1 locus. We also examined the same samples for inactivation of the RB1 gene by immunohistochemical analysis of paraffin‐embedded samples, but no significant variation in RB protein expression was detected. In addition, we also performed PCR‐single‐strand conformational polymorphism (SSCP) analysis to detect any mutation of the RB1 gene using 52 primer pairs, which covers all exons of this gene. We found no mutations of the RB1 gene in our samples. Interestingly, we found significant correlation between LOH of 13q14.3 and lymph node metastasis. Our results indicate that LOH of 13q is a common event in oncogenesis and/or progression of oral SCC, and also suggest the existence of a new suppressor gene near D13S273‐D13S176 loci which may play a role in these events. Int. J. Cancer (Pred. Oncol.) 79:312–317, 1998.

MicroRNA-125b regulates proliferation and radioresistance of oral squamous cell carcinoma.

Background:MicroRNAs (miRNAs) are involved in essential biological activities, and have been reported to exhibit differential expression profiles in various cancers. Our previous study demonstrated that intercellular adhesion molecule-2 (ICAM2) inhibition induces radiosensitisation in oral squamous cell carcinoma (OSCC) cells. Thus, we hypothesised that certain miRNAs play crucial roles in radioresistance in OSCC by regulating ICAM2 expression.Methods:Because predicted target gene analyses revealed that microRNA-125b (miR-125b) potentially regulates ICAM2 mRNA expression, we examined the association between miR-125b and radioresistance. The expression of miR-125b was investigated by real-time quantitative reverse transcriptase–PCR. For a functional analysis, miR-125b was transfected to OSCC-derived cells.Results:A downregulated expression of miR-125b was found in OSCC-derived cell lines and OSCC samples. The miR-125b-transfected cells showed a decreased proliferation rate, enhanced radiosensitivity to X-ray irradiation and diminished ICAM2 mRNA expression. Moreover, miR-125b expression correlated with OSCC tumour staging and survival.Conclusion:These findings suggested that the downregulated miR-125b expression was associated with proliferation and radioresistance mechanisms, probably through ICAM2 signalling. Thus, controlling the expression or activity of miR-125b might contribute to suppressing proliferation and overcoming radioresistance in OSCC.

Epithelial cell transforming sequence 2 in human oral cancer.

Background Epithelial cell transforming sequence 2 (ECT2) is a guanine nucleotide exchange factor for Rho family GTPase, which has been implicated in the malignant phenotype of human cancers. Little is known about the effect of a high level of ECT2 in regulating oral cancer cell behavior. In this study, we investigated the involvement of ECT2 in oral squamous cell carcinoma (OSCC). Methodology/Principal Findings We analyzed ECT2 expression in OSCC-derived cell lines and primary OSCCs compared with matched normal tissue (n = 96) by quantitative reverse transcriptase-polymerase chain reaction, Western blot, and immunohistochemistry. We then evaluated the correlation between the ECT2 expression status in primary OSCCs and the clinicopathological features. ECT2 expression was significantly up-regulated in OSCCs in vitro and in vivo (p<0.05). Among the clinical variables analyzed, higher ECT2 expression also was associated with the TNM stage grading (p<0.05). When we performed functional analyses of ECT2 in OSCC-derived cells using the shRNA system, the cellular proliferation of the ECT2 knockdown cells decreased significantly compared with the control cells (p<0.05). Cell cycle analysis by flow cytometry showed arrest of cell cycle progression at the G1 phase in the ECT2 knockdown cells. We also found up-regulation of the Cip/Kip family of the cyclin-dependent kinase inhibitors, p21cip1 and p27kip1, and down-regulation of cyclin D1, cyclin E, and CDK4. These data suggested that the elevated Cip/Kip family induced inhibition of the cyclin D1-CDK complex activity leading to cell cycle arrest at the G1 phase. Conclusions/Significance Our results proposed for the first time that ECT2 is an indicator of cellular proliferation in OSCCs and that ECT2 might be a potential therapeutic target for the development of new treatments for OSCCs.

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.

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.

Dermatopontin: a potential predictor for metastasis of human oral cancer.

Dermatopontin (DPT), a component of the extracellular matrix (ECM), is involved in promotion of cellular adhesion and ECM assembly activities. However, the role of DPT in the pathogenesis of carcinoma is unclear. We evaluated DPT expression in human oral cancer and its possible roles including cellular adhesion and invasiveness. We first investigated the DPT mRNA and protein expression status in human oral squamous cell carcinoma (OSCC)‐derived cells. Real‐time quantitative reverse transcriptase‐polymerase chain reaction (qRT‐PCR) and immunoblotting analysis detected frequent downregulation of DPT in OSCC‐derived cells compared to human normal oral keratinocytes. To assess the epigenetic regulation of DPT, OSCC‐derived cells were treated with a histone deacetylase inhibitor, sodium butyrate (NaB). NaB restored the DPT expression in OSCC‐derived cells. DPT‐overexpressed cells were examined whether DPT could contribute to cellular adhesion and invasiveness. Markedly, increased adhesion and decreased invasiveness in DPT‐overexpressed cells were found compared to mock‐transfected cells. Adhesion of DPT‐overexpressed cells was inhibited by α3β1 integrin functional blocking antibody. OSCC‐derived cells treated with NaB also decreased invasiveness. The expression status of DPT in primary OSCCs (n = 97) was analyzed and compared to clinicopathological behavior. DPT expression in primary OSCCs was significantly lower (p < 0.05) than in the normal counterparts and was correlated significantly (p < 0.05) with regional lymph node metastasis. Our data provided strong evidence that downregulation of DPT is a characteristic event in OSCCs and that DPT was correlated with cellular adhesion and invasiveness. Therefore, DPT might play an important role in regulating tumor invasion and metastasis.

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.