Oral Leukoplakia Tissues Research Articles

Abstract 430: Identification of novel methylation-silenced genes in oral squamous cell carcinomas

Abstract To identify novel methylation-silenced genes in oral squamous cell carcinomas (OSCCs), we took a combined approach of methylated DNA immunoprecipitation (MeDIP)-CpG island (CGI) microarray analysis and expression microarray analysis after treatment with a demethylating agent. MeDIP-CGI microarray revealed that promoter CGIs of 324 genes were methylated in two OSCC cell lines (Ca9-22, HSC-2). By treatment of the cell lines with 5-aza-2′-deoxycytidine, 125 of the 324 genes were up-regulated in at least one of the two cell lines. For 23 of the 125 genes that have not been focused on in the field of oral malignancy, we analyzed their promoter methylation statuses in 16 normal oral tissues, five OSCC cell lines, 26 OSCC tissues and 24 oral leukoplakia tissues. Ten promoter CGIs were methylated not only in OSCC cell lines but also OSCC tissues. The methylation profile indicated the presence of the CpG island methylator phenotype (CIMP) in OSCC tissues. Furthermore, the CIMP was observed in three of the 13 oral dysplastic leukoplakia tissues. The CIMP was considered as one of early molecular events in malignant transformation from oral premalignant lesions to OSCCs. Citation Format: Masanobu Abe, Satoshi Yamashita, Yoshiyuki Mori, Takahiro Abe, Hideto Saijo, Toshikazu Ushijima, Tsuyoshi Takato. Identification of novel methylation-silenced genes in oral squamous cell carcinomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 430. doi:10.1158/1538-7445.AM2014-430

Endoscopic swept-source optical coherence tomography based on a two-axis microelectromechanical system mirror

A microelectromechanical system (MEMS) mirror based endoscopic swept-source optical coherence tomography (SS-OCT) system that can perform three-dimensional (3-D) imaging at high speed is reported. The key component enabling 3-D endoscopic imaging is a two-axis MEMS scanning mirror which has a 0.8×0.8 mm2 mirror plate and a 1.6×1.4 mm2 device footprint. The diameter of the endoscopic probe is only 3.5 mm. The imaging rate of the SS-OCT system is 50 frames/s. OCT images of both human suspicious oral leukoplakia tissue and normal buccal mucosa were taken in vivo and compared. The OCT imaging result agrees well with the histopathological analysis.

Identification of genes related to carcinogenesis of oral leukoplakia by oligo cancer microarray analysis

The aim of this study was to identify genes that are predictive of carcinogenic change in patients of oral leukoplakia (OLK) using a cancer-related microarray. Candidate biomarkers were discovered using the Oligo GEArray OHS-802 and validated on independent samples by semi-quantitative reverse transcription-PCR (RT-PCR) and real-time RT-PCR. Both the discovery and the validation cohorts of samples included normal oral tissues, OLK tissues and oral squamous cell carcinoma (OSCC) tissues. Based on the microarray results, we found that there were nine genes successively up-regulated or down-regulated more than 2-fold between the normal group and OLK group and then again between the OLK group and OSCC group. The expression levels of the nine signature genes had statistically significant differences (p<0.05) between the OLK and normal groups and between the OSCC and OLK groups. In summary, the expression of the 9 signature genes might be representative of OLK carcinogenesis. A cancer-related microarray was used to identify a panel of candidate biomarkers for determining carcinogenesis of OLK lesions, in combination with semi-quantitative and real-time RT-PCR to validate the results. Our data indicated that alterations in gene expression that result in carcinogenesis can be identified in precancerous oral tissues.

Immunohistochemical detection of early-stage carcinogenesis of oral leukoplakia by increased DNA-instability and various malignancy markers.

The degree of DNA instability as determined by immunohistochemical staining with anti-single-stranded DNA antibody after acid hydrolysis (the DNA instability test) was used as a marker of malignancy. The test was applied to tissues of oral leukoplakia assessed histopathologically as hyperplasia (38 cases), mild (12 cases), moderate (11 cases) and severe (8 cases) dysplasia, and invasive squamous cell carcinoma (SCC, 20 cases). Tissues were subjected to immunohistochemical staining for proliferating cell nuclear antigen (PCNA), p53, DNA-fragmentation factor 45 (DFF45), analysis of various AgNORs parameters, and triple immunostaining for vascular endothelial growth factor (VEGF), CD34, and PCNA. The DNA instability test was positive in 20 (100%) SCC cases, 8 (100%) severe dysplasia cases, 8 (72.7%) moderate dysplasia cases, 6 (50.0%) mild dysplasia cases, and 9 (23.7%) hyperplasia cases, indicating malignancy. The proportion of lesions positive for PCNA, p53, DFF45, and values of AgNORs parameters steadily increased from hyperplasia to mild, moderate and severe dysplasia, and SCC, especially in those showing positive DNA instability test, indicative of malignancy. Based on these results, 44.9% of leukoplakia were malignant tissues, namely carcinoma in situ. The proportion of PCNA-positive vascular endothelial cells in the vicinity of VEGF-positive epithelial lesion was significantly higher than that of negative DNA instability lesions, as revealed by immunohistochemical triple staining for VEGF, CD34, and PCNA. Our results suggest that increased DNA instability, enhanced proliferative activity, p53 mutation, and induction of DFF45 and VEGF may allow cancer cell proliferation, enhance their survival by escaping apoptosis, and provide abundant nutrients during early-stage carcinogenesis of oral leukoplakia.

Involvement of potential pathways in malignant transformation from Oral Leukoplakia to Oral Squamous Cell Carcinoma revealed by proteomic analysis

BackgroundOral squamous cell carcinoma (OSCC) is one of the most common forms of cancer associated with the presence of precancerous oral leukoplakia. Given the poor prognosis associated with oral leukoplakia, and the difficulties in distinguishing it from cancer lesions, there is an urgent need to elucidate the molecular determinants and critical signal pathways underlying the malignant transformation of precancerous to cancerous tissue, and thus to identify novel diagnostic and therapeutic target.ResultsWe have utilized two dimensional electrophoresis (2-DE) followed by ESI-Q-TOF-LC-MS/MS to identify proteins differentially expressed in six pairs of oral leukoplakia tissues with dysplasia and oral squamous cancer tissues, each pair was collected from a single patient. Approximately 85 differentially and constantly expressed proteins (> two-fold change, P < 0.05) were identified, including 52 up-regulated and 33 down-regulated. Gene ontological methods were employed to identify the biological processes that were over-represented in this carcinogenic stage. Biological networks were also constructed to reveal the potential links between those protein candidates. Among them, three homologs of proteosome activator PA28 a, b and g were shown to have up-regulated mRNA levels in OSCC cells relative to oral keratinocytes.ConclusionVarying levels of differentially expressed proteins were possibly involved in the malignant transformation of oral leukoplakia. Their expression levels, bioprocess, and interaction networks were analyzed using a bioinformatics approach. This study shows that the three homologs of PA28 may play an important role in malignant transformation and is an example of a systematic biology study, in which functional proteomics were constructed to help to elucidate mechanistic aspects and potential involvement of proteins. Our results provide new insights into the pathogenesis of oral cancer. These differentially expressed proteins may have utility as useful candidate markers of OSCC.

Increased expression of galectin-1 is associated with human oral squamous cell carcinoma development

Galectin-1 (Gal-1) is a newly found immunoregulatory carbohydrate-binding protein in cancer biology. The purpose of this study was to evaluate the effects of Gal-1 on oral squamous cell carcinoma (OSCC) development. Immunohistochemistry, Western blotting and RT-PCR were carried out in 62 primary OSCC, 38 oral leukoplakia (OPL) tissues to detect the Gal-1 expression in both protein and mRNA levels. Ten normal oral mucosa (NOM) tissues were used as control. Gal-1 protein was significantly overexpressed in OSCC cancer cells and OPL prickle cells compared to NOM (P<0.05). In accordance with Gal-1 protein, Gal-1 mRNA was also up-regulated in OSCC tissues and OPL tissues. Furthermore, both the Gal-1 protein and mRNA in OSCC tissues were higher than in OPL tissues (P<0.05). Our data supports the important roles of Gal-1 in OSCC development and suggests that Gal-1 upregulation in the OSCC and OPL tissues might be a predictor of early oral carcinogenesis.

Antitumor Effect of Retinoic Acid Receptor-β2 Associated with Suppression of Cyclooxygenase-2

Retinoic acid receptor-beta2 (RAR-beta2) is a putative tumor suppressor gene in various cancers. To determine the underlying molecular mechanisms, we transfected RAR-beta2 cDNA into esophageal cancer TE-1 and TE-8 cells and found that RAR-beta2 suppressed tumor cell growth in vitro and tumor formation in nude mice in TE-8 cells, whereas the stable transfection of RAR-beta2 did not restore retinoid sensitivity or inhibit tumor formation in nude mouse in TE-1 cells. Molecularly, we revealed that RAR-beta2 antitumor activity was associated with expression and suppression of cyclooxygenase-2 (COX-2) in these tumor cell lines. Moreover, antisense RAR-beta2 cDNA induced COX-2 expression in TE-3 cells. Furthermore, when COX-2 expression is first blocked by using antisense COX-2 expression vector, the effect of RAR-beta2 is diminished in these tumor cells. In addition, we analyzed expression of RAR-beta2 and COX-2 mRNA in tissue specimens and found that RAR-beta2 expression is associated with low levels of COX-2 expression in esophageal cancer tissues. Induction of RAR-beta2 expression in oral leukoplakia tissues after the patients treated with 13-cis RA correlated with a reduction in COX-2 expression and clinical response. Our findings indicate that some of RAR-beta2 antitumor activities are mediated by suppression of COX-2 expression in some of these esophageal cancer cells. After correlating antitumor effect of RAR-beta2 with COX-2 expression in the published studies, we also found the association. Thus, further studies will determine whether manipulation of COX-2 expression in different cancers can antagonize RAR-beta2 activity.

Amplification methods of DNA from paraffin-embedded tissues of oral leukoplakia

To set up a feasible method for detection of objective genes from paraffin-embedded tissues of oral leukoplakia (OLK). Twenty-five pieces of formalin-fixed, paraffin-embedded tissues of OSCC were selected and ATM gene was detected respectively by 3 methods: the microdissection-nested PCR method, proteinase K-PCR method and conventional phenol-chloroform-PCR method. The positivity rates were compared statistically. The positivity rates of these 3 methods were 84%, 52% and 64% respectively. Significant difference was found in positivity rate between the microdissection-nested PCR method and the proteinase K-PCR method (P = 0.032). The microdissection-nested PCR method merits recommendation because it is more efficient, easy to perform and has the advantage of less sample amount.

Interphase nucleolar organizer region distribution in the tissues of oral leukoplakia, oral submucous fibrosis and oral cancer

The AgNOR technique, was applied to oral tissue sections of 185 oral cancer, 42 oral leukoplakia, 37 oral submucous fibrosis and 10 normal subjects to investigate whether any correlation held good in these different tissues. Compared to the AgNOR counts in normal oral epithelium, there was a gradation in increase in the mean AgNOR counts from oral leukoplakia to oral submucous fibrosis to oral carcinoma (P<0.01). This suggests that AgNOR count parallels with the degree of neoplastic transformation of oral epithelium. Three oral submucous fibrosis patients who showed very high AgNOR counts as that of oral cancer patients, later developed oral carcinoma. Among the oral cancer tissues, the moderately and poorly differentiated subtypes showed higher AgNOR counts and scattered distribution pattern than the well differentiated subtype which showed a clustered distribution pattern. These results suggest that AgNOR technique can be utilised as a diagnostic and prognostic indicator in premalignant and malignant oral tissues.

C-Ha-ras oncogene in oral leukoplakia tissues.

The amplification and the G-T mutation at codon 12 of the C-Ha-ras oncogene in oral leukoplakia tissues were analyzed by using polymerase chain reaction (PCR) and molecular biologic technique. The results showed that these tissues had no amplification of Ha-ras oncogene. Only one case harbored G-T mutation in 11 oral leukoplakias, but this mutation was absent in 10 normal oral mucosal tissues. The possible role and significance of C-Ha-ras oncogene in oral precancerous lesions were also discussed.