Katsunori Ogawara

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.

Evidence for two distinct tumor-suppressor gene loci on the long arm of chromosome 11 in human oral cancer.

Loss of heterozygosity (LOH) on human chromosome 11 has been reported in a variety of human cancers. To search for the existence of tumor‐suppressor gene(s) associated with oral squamous cell carcinoma (SCC) on chromosome 11, we have performed high‐resolution deletion mapping in 31 patients with oral SCC using 22 microsatellite markers for this chromosomal region. LOH was observed in 14 of 25 cases (56.0%) that were informative with at least one locus. Most allelic deletions detected in our study were specific to the long arm of the chromosome. Furthermore, the data presented here show 2 distinct, commonly deleted regions. The first region, with frequent LOH, was restricted between markers D11S939 and D11S924 separated by 3 centimorgans (cM) on chromosome 11q23. The second region of common deletion was identified between markers D115912 and D11S910, separated by 7 cM at 11q25. Our results suggest that at least 2 tumor‐suppressor genes involved in the development of oral SCC are present on the long arm of chromosome II.

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.

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

BackgroundGelsolin-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.MethodsMatched 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 Fishers exact test. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was used to estimate CapG expression at the mRNA level.ResultsIn 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.ConclusionOur 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.

Overexpression of CDCA2 in Human Squamous Cell Carcinoma: Correlation with Prevention of G1 Phase Arrest and Apoptosis

Cell division cycle associated 2 (CDCA2) recruits protein phosphatase 1 to chromatin to antagonize activation of ataxia telangiectasia mutated (ATM)-dependent signal transduction. ATM kinase plays a critical role in the DNA damage response and its phosphorylation cascade to inhibit the p53-MDM2 interaction, which releases p53 to induce p21 and G1 cell-cycle arrest. However, the relevance of CDCA2 to human malignancy including oral squamous cell carcinoma (OSCC) is unknown. In the current study, we found that CDCA2 expression was up-regulated in OSCC cell lines. Functional studies with shRNA system showed that knockdown of CDCA2 significantly (P<0.05) inhibited cellular proliferation compared with the control cells by arresting cell-cycle progression at the G1 phase and up-regulating the cyclin-dependent kinase inhibitors (p21Cip1, p27Kip1, p15INK4B, and p16INK4A). CDCA2 knockdown also promoted apoptosis after treatment with the DNA damage reagent, cisplatin. In clinical samples, the CDCA2 protein expression level in primary OSCCs was significantly (P<0.05) greater than in matched normal oral tissues (67/85, 79%). Furthermore, CDCA2-positive cases were correlated significantly (P<0.05) with high cancer progression. Our results showed for the first time that CDCA2 frequently is overexpressed in OSCCs and might be associated closely with OSCC progression by preventing cell-cycle arrest and apoptosis.

Overexpression of cell cycle regulator CDCA3 promotes oral cancer progression by enhancing cell proliferation with prevention of G1 phase arrest

BackgroundCell division cycle associated 3 (CDCA3), part of the Skp1-cullin-F-box (SCF) ubiquitin ligase, refers to a trigger of mitotic entry and mediates destruction of the mitosis inhibitory kinase. Little is known about the relevance of CDCA3 to human malignancy including oral squamous cell carcinoma (OSCC). We aimed to characterize the expression state and function of CDCA3 in OSCC.MethodsWe evaluated CDCA3 mRNA and protein expression in both OSCC-derived cell lines and primary OSCCs and performed functional analyses of CDCA3 in OSCC-derived cells using the shRNA system.ResultsThe CDCA3 expression at both the mRNA and protein levels was frequently up-regulated in all cell lines examined and primary tumors (mRNA, 51/69, 74 %; protein, 79/95, 83 %) compared to normal controls (p < 0.001). In contrast, no significant level of CDCA3 protein expression was seen in oral premalignant lesions (OPLs) (n = 20) compared with the expression in OSCCs. Among the clinical variables analyzed, the CDCA3 expression status was closely related to tumor size (p < 0.05). In addition, suppression of CDCA3 expression with shRNA significantly (p < 0.05) inhibited cellular proliferation compared with the control cells by arresting cell-cycle progression at the G1 phase. Further, there was up-regulation of the cyclin-dependent kinase inhibitors (p21Cip1, p27Kip1, p15INK4B, and p16INK4A) in the knockdown cells.ConclusionThe current results showed that overexpression of CDCA3 occurs frequently during oral carcinogenesis and this overexpression might be associated closely with progression of OSCCs by preventing the arrest of cell-cycle progression at the G1 phase via decreased expression of the cyclin-dependent kinase inhibitors.

State of heat shock factor 1 expression as a putative diagnostic marker for oral squamous cell carcinoma

Heat shock factor 1 (HSF1) is responsible for expres-- sion of a large class of heat shock proteins that have been implicated in the malignant phenotype of human cancers. Little is known about the effect of a high level of HSF1 on the behavior of oral squamous cell carcinoma (OSCC). In this study, we assessed the value of HSF1 for predicting clinical outcomes in OSCC. Quantitative reverse transcriptase-polymerase chain reaction and Western blotting showed that the expressions of HSF1 mRNA and protein in OSCC-derived cell lines (HSC-2, HSC-3, HSC-4, Sa3, Ca9-22, KON and Ho-1-u-1) were elevated compared with those in human normal oral keratinocytes (P<0.05). Similar to in vitro data, HSF1 mRNA expression in primary OSCCs (n=50) was significantly greater than in normal counterparts (P<0.05). Since HSF1 was observed in the nucleus and cytoplasm by immu-- nohistochemistry, we investigated the correlation between the HSF1 expression status at each subcellular location and the clinical behavior of OSCCs. Among the clinical classifications, higher nuclear HSF1 expression was closely related to tumor size and histopathologic types (P<0.05). These results showed for the first time that nuclear HSF1 expression may contribute to cancer progression and that HSF1 might be a potential diagnostic biomarker and a therapeutic target for OSCCs.