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1.  Gene expression profiling identifies genes predictive of oral squamous cell carcinoma 
Oral squamous cell carcinoma (OSCC) is associated with substantial mortality and morbidity. To identify potential biomarkers for early detection of invasive OSCC, we compared gene expression of incident primary OSCC, oral dysplasia, and clinically normal oral tissue from surgical patients without head and neck cancer or pre-neoplastic oral lesions (controls), using Affymetrix U133 2.0 Plus arrays. We identified 131 differentially expressed probe sets using a training set of 119 OSCC patients and 35 controls. Forward and stepwise logistic regression analyses identified 10 successive combinations of genes which expression differentiated OSCC from controls. The best model included LAMC2, encoding laminin gamma 2 chain, and COL4A1, encoding collagen, type IV, alpha 1 chain. Subsequent modeling without these two markers showed that COL1A1, encoding collagen, type I, alpha 1 chain, and PADI1, encoding peptidyl arginine deiminase, type 1, also can distinguish OSCC from controls. We validated these two models using an internal independent testing set of 48 invasive OSCC and 10 controls and an external testing set of 42 head and neck squamous cell carcinoma (HNSCC) cases and 14 controls (GEO GSE6791), with sensitivity and specificity above 95%. These two models were also able to distinguish dysplasia (n=17) from control (n=35) tissue. Differential expression of these four genes was confirmed by qRT-PCR. If confirmed in larger studies, the proposed models may hold promise for monitoring local recurrence at surgical margins and the development of second primary oral cancer in OSCC patients.
doi:10.1158/1055-9965.EPI-07-2893
PMCID: PMC2575803  PMID: 18669583
oral squamous cell carcinoma; oral cancer; genetic expression profiles; microarrays
2.  Genome-wide gene expression profiles of HPV-positive and HPV-negative oropharyngeal cancer: potential implications for treatment choices 
Objective
To study the difference in gene expression between human papillomavirus (HPV)-positive and HPV-negative oral squamous cell carcinoma (OSCC).
Design
We used Affymetrix U133 plus 2.0 arrays to examine gene expression profiles of OSCC and normal oral tissue. HPV DNA was detected using PCR followed by the Roche Linear Array HPV Genotyping Test, and the differentially expressed genes were analyzed to examine their potential biological roles using the Ingenuity Pathway Analysis Software (IPA 5.0).
Subjects
Tumor tissue from 119 primary OSCC patients and normal oral tissue from 35 patients without cancer, all of whom were treated at three University of Washington-affiliated medical centers.
Results
HPV DNA was found in 41 of 119 (34.5%) tumors and 2 of 35 (5.7%) normal tissue samples, with 39 of 43 HPV being HPV type 16; there was a higher prevalence of HPV DNA in oropharyngeal cancer (23 of 31) than in oral cavity cancer (18 of 88). We found no significant difference in gene expression between HPV-positive and HPV-negative oral cavity cancer but found 446 probe sets (347 known genes) differentially expressed between HPV-positive and HPV-negative oropharyngeal cancer. The most prominent functions of these genes are DNA replication, DNA repair, and cell cycle. Some genes differentially expressed between HPV-positive and HPV-negative oropharyngeal cancer (e.g., TYMS, STMN1, CCND1 and RBBP4) are involved in chemotherapy or radiation sensitivity.
Conclusion
These results suggest that differences in the biology of HPV-positive and HPV-negative oropharyngeal cancer may have implications for the management of patients with these different tumors.
doi:10.1001/archoto.2008.540
PMCID: PMC2761829  PMID: 19221247
Human papillomavirus; oral cancer; gene expression
3.  Expression of Angiopoietin-2 and Vascular Endothelial Growth Factor Receptor-3 Correlates with Lymphangiogenesis and Angiogenesis and Affects Survival of Oral Squamous Cell Carcinoma 
PLoS ONE  2013;8(9):e75388.
Background
Both Ang-2 and VEGFR-3 are major regulators of angiogenesis and lymphangiogenesis, respectively, and thus may affect prognosis of OSCC. We sought to determine the associations between Ang-2 and VEGFR-3 expression and survival of OSCC.
Methods
Ang-2 and VEGFR-3 expression was determined immunohistochemically in tumor tissues from 112 patients with OSCC; OSCC-adjacent noncancerous oral tissue from 85 OSCC patients; and normal oral mucosa from 37 cancer-free individuals. A log-rank test and Cox proportional hazard models were used to compare survival among different groups with expression of Ang-2 and VEGFR-3.
Results
Ang-2 and VEGFR-3 expression was upregulated in OSCC compared to nontumor tissue (all P<0.05). High Ang-2 expression positively correlated with microvessel density (MVD) (P<0.01), and high VEGFR-3 expression positively correlated with lymph node metastasis (P<0.01) and lymphatic vessel density (LVD) (P<0.01). The patients with high expression of Ang-2 alone or in combination with VEGFR-3 had a significantly worse survival than in patients with low expression of Ang-2 or any other co-expression status (all P<0.05), respectively. Furthermore, multivariable analysis showed that patients with high expression of Ang-2 alone or in combination with VEGFR-3 had a significantly increased risk of death compared with those with low expression of Ang-2 or any other co-expression status (HR, 2.7, 95% CI, 1.1–6.2 and 5.0, 1.3–15.4, respectively).
Conclusions
These results suggest that increased expression in tumors of Ang-2 may individually, or in combination with VEGFR-3, predict poor prognosis of OSCC.
doi:10.1371/journal.pone.0075388
PMCID: PMC3770542  PMID: 24040410
4.  Clinical Significance of Keap1 and Nrf2 in Oral Squamous Cell Carcinoma 
PLoS ONE  2013;8(12):e83479.
Oxidative stress has been reported to play an important role in progression and prognostication in various kinds of cancers. However, the role and clinical significance of oxidative stress markers Keap1 and Nrf2 in oral squamous cell carcinoma (OSCC) has not been elucidated. This study aimed to investigate the correlation of oxidative stress markers Keap1 and Nrf2 expression and pathological features in OSCC by using tissue microarray. Tissue microarrays containing 17 normal oral mucosa, 7 oral epithelial dysplasia and 43 OSCC specimens were studied by immunohistochemistry. The association among these proteins and pathological features were analyzed. Expression of oxidative stress markers Keap1, Nrf2, and antioxidants PPIA, Prdx6, as well as CD147 was found to increase consecutively from normal oral mucosa to OSCC, and the Keap1, Nrf2, PPIA, Prdx6, CD147 expression in OSCC were significantly higher when compared to normal oral mucosa. Expression of Keap1, Nrf2 in tumors was not found to be significantly associated with T category, lymph node metastases, and pathological grade. Furthermore, we checked the relationship among these oxidative stress markers and found that Keap1 was significantly correlated with Nrf2, Prdx6 and CD147. Significant relationship between Nrf2 and Prdx6 was also detected. Finally, we found patients with overexpression of Keap1 and Nrf2 had not significantly worse overall survival by Kaplan–Meier analysis. These findings suggest that ROS markers are associated with carcinogenesis and progression of OSCC, which may have prognostic value and could be regarded as potential therapeutic targets in OSCC.
doi:10.1371/journal.pone.0083479
PMCID: PMC3873935  PMID: 24386210
5.  PITX1 is a reliable biomarker for predicting prognosis in patients with oral epithelial dysplasia 
Oncology Letters  2013;7(3):750-754.
Paired-like homeodomain 1 (PITX1) genes are essential in human development. In the present study, PITX1 protein expression was evaluated in human normal oral mucosa, oral epithelial dysplasia and oral squamous cell carcinoma (OSCC), with the aim of examining the expression patterns of these critical genes during the multi-stage transformation of oral epithelial dysplasia to OSCC. PITX1 and Ki-67 expression were assessed by immunohistochemistry in 26 individuals with normal oral mucosa, 106 patients with oral epithelial dysplasia and 97 OSCC patients. The labeling indices (LIs) of PITX1 and Ki-67 were calculated and their correlation with the incidence of malignancy was evaluated. The PITX1 LI of the dysplasia specimens was significantly lower than that of the normal oral mucosa samples, but significantly higher than that of the OSCC samples. The oral epithelial dysplasia patients that exhibited low PITX1 expression showed a significantly higher incidence of malignant transformation than those exhibiting high PITX1 expression, regardless of the histological grades of their oral epithelial dysplasias. On the other hand, no correlation was observed between the Ki-67 LI and the incidence of malignancy. These results suggested that PITX1 suppression is associated with malignant transformation in the oral epithelium and that PITX1 expression may serve as a novel biomarker for predicting prognosis in oral epithelial dysplasia.
doi:10.3892/ol.2013.1775
PMCID: PMC3919858  PMID: 24527083
PITX1; oral epithelial dysplasia; immunohistochemistry; biomarker
6.  Activation of MAP Kinase Signaling Through ERK5 But Not ERK1 Expression Is Associated with Lymph Node Metastases in Oral Squamous Cell Carcinoma (OSCC)1,2 
Neoplasia (New York, N.Y.)  2008;10(5):462-470.
In an attempt to further elucidate the pathomechanisms in oral squamous cell carcinoma (OSCC), gene expression profiling was performed using a whole-transcriptome chip that contains 35,035 gene-specific 70mere oligonucleotides (Human OligoSet 4.0; Operon, Cologne, Germany) to a set of 35 primary OSCCs. Altogether, 7390 genes were found differentially expressed between OSCC tumor samples and oral mucosa. To characterize the major biologic processes in this tumor collection, MAPPFinder, a component of GenMAPP version 2.1, was applied to this data set to generate a statistically ranked list of molecular signaling pathways. Among others, cancer-related pathways, such as mitogen-activated protein (MAP) kinase signaling (z score = 4.6, P < .001), transforming growth factor-beta signaling (z score = 3.0, P = .015), and signaling pathways involved in apoptosis (z score = 2.1, P = .037), were found deregulated in the OSCC collection analyzed. Focusing on the MAP kinase signaling pathway, subsequent tissue microarray analyses by immunohistochemistry revealed an increase in protein expression of MAP kinase-related proteins ERK1 in 22.8% (48 of 209) and ERK5 in 27.4% (76 of 277), respectively. An association of high ERK5 but not of high ERK1 expression with advanced tumor stage and the presence of lymph node metastases was found (P = .008 and P = .016, respectively). Our analysis demonstrates the reliability of the combined approach of gene expression profiling, signaling pathway analyses, and tissue microarray analysis to detect novel distinct molecular aberrations in OSCC.
PMCID: PMC2373911  PMID: 18472963
7.  Involvement of TSC genes and differential expression of other members of the mTOR signaling pathway in oral squamous cell carcinoma 
BMC Cancer  2008;8:163.
Background
Despite extensive research, the five-year survival rate of oral squamous cell carcinoma (OSCC) patients has not improved. Effective treatment of OSCC requires the identification of molecular targets and signaling pathways to design appropriate therapeutic strategies. Several genes from the mTOR signaling pathway are known to be dysregulated in a wide spectrum of cancers. However, not much is known about the involvement of this pathway in tumorigenesis of OSCC. We therefore investigated the role of the tumor suppressor genes, TSC1 and TSC2, and other members of this pathway in tumorigenesis of OSCC.
Methods
Expression of genes at the RNA and protein levels was examined by semi-quantitative RT-PCR and western blot analyses, respectively. Loss of heterozygosity was studied using matched blood and tumor DNA samples and microsatellite markers from the TSC1, TSC2 and PTEN candidate regions. The effect of promoter methylation on TSC gene expression was studied by treating cells with methyltransferase inhibitor 5-azacytidine. Methylation status of the TSC2 promoter in tissue samples was examined by combined bisulfite restriction analysis (COBRA).
Results
The semi-quantitative RT-PCR analysis showed downregulation of TSC1, TSC2, EIF4EBP1 and PTEN, and upregulation of PIK3C2A, AKT1, PDPK1, RHEB, FRAP1, RPS6KB1, EIF4E and RPS6 in tumors. A similar observation was made for AKT1 and RPS6KB1 expression in tumors at the protein level. Investigation of the mechanism of downregulation of TSC genes identified LOH in 36.96% and 39.13% of the tumors at the TSC1 and TSC2 loci, respectively. No mutation was found in TSC genes. A low LOH rate of 13% was observed at the PTEN locus. Treatment of an OSCC cell line with the methyltransferase inhibitor 5-azacytidine showed a significant increase in the expression of TSC genes, suggesting methylation of their promoters. However, the 5-azacytidine treatment of non-OSCC HeLa cells showed a significant increase in the expression of the TSC2 gene only. In order to confirm the results in patient tumor samples, the methylation status of the TSC2 gene promoter was examined by COBRA. The results suggested promoter hypermethylation as an important mechanism for its downregulation. No correlation was found between the presence or absence of LOH at the TSC1 and TSC2 loci in 50 primary tumors to their clinicopathological variables such as age, sex, T classification, stage, grade, histology, tobacco habits and lymph node metastasis.
Conclusion
Our study suggests the involvement of TSC genes and other members of the mTOR signaling pathway in the pathogenesis of OSCC. LOH and promoter methylation are two important mechanisms for downregulation of TSC genes. We suggest that known inhibitors of this pathway could be evaluated for the treatment of OSCC.
doi:10.1186/1471-2407-8-163
PMCID: PMC2430211  PMID: 18538015
8.  Evaluation of PTEN Immunoexpression in Oral Submucous Fibrosis: Role in Pathogenesis and Malignant Transformation 
Head and Neck Pathology  2012;6(3):314-321.
Oral submucous fibrosis (OSMF) is a chronic debilitating disease and a premalignant condition of the oral cavity characterized by generalized submucosal fibrosis. Despite its precancerous nature, the molecular biology regarding its malignant potential has not been extensively studied. PTEN, a known tumor suppressor gene is mutated in a majority of human cancers and has also been implicated in several fibrotic disorders. The present study aims to evaluate the expression of PTEN in OSMF and oral squamous cell carcinoma (OSCC) and correlate it with the pathogenesis and malignant transformation of OSMF. 60 cases total of OSMF (30) and OSCC (30) were subjected to immunohistochemistry using PTEN antibody. Ten normal oral mucosa (NOM) specimens were also stained as controls. There was progressive loss of PTEN expression from normal mucosa to OSMF and OSCC (p ≤ 0.001). Significant differences were observed for PTEN expression between NOM and OSMF, OSMF and OSCC as well as NOM and OSCC. Though a progressive loss of PTEN was noticed between early OSMF and advanced OSMF, the variation did not reach statistical significance (p ≥ 0.001). Data suggest that there is a significant loss of PTEN expression in OSMF as compared to normal oral mucosa and that this trend increased from OSMF to OSCC. Thus, alteration of PTEN is likely an important molecular event in OSMF pathogenesis and oral carcinogenesis.
Electronic supplementary material
The online version of this article (doi:10.1007/s12105-012-0341-z) contains supplementary material, which is available to authorized users.
doi:10.1007/s12105-012-0341-z
PMCID: PMC3422583  PMID: 22392409
PTEN; Oral submucous fibrosis; Oral squamous cell carcinoma; Carcinogenesis; Pathogenesis; Malignant transformation
9.  Integrative Genomics in Combination with RNA Interference Identifies Prognostic and Functionally Relevant Gene Targets for Oral Squamous Cell Carcinoma 
PLoS Genetics  2013;9(1):e1003169.
In oral squamous cell carcinoma (OSCC), metastasis to lymph nodes is associated with a 50% reduction in 5-year survival. To identify a metastatic gene set based on DNA copy number abnormalities (CNAs) of differentially expressed genes, we compared DNA and RNA of OSCC cells laser-microdissected from non-metastatic primary tumors (n = 17) with those from lymph node metastases (n = 20), using Affymetrix 250K Nsp single-nucleotide polymorphism (SNP) arrays and U133 Plus 2.0 arrays, respectively. With a false discovery rate (FDR)<5%, 1988 transcripts were found to be differentially expressed between primary and metastatic OSCC. Of these, 114 were found to have a significant correlation between DNA copy number and gene expression (FDR<0.01). Among these 114 correlated transcripts, the corresponding genomic regions of each of 95 transcripts had CNAs differences between primary and metastatic OSCC (FDR<0.01). Using an independent dataset of 133 patients, multivariable analysis showed that the OSCC–specific and overall mortality hazards ratio (HR) for patients carrying the 95-transcript signature were 4.75 (95% CI: 2.03–11.11) and 3.45 (95% CI: 1.84–6.50), respectively. To determine the degree by which these genes impact cell survival, we compared the growth of five OSCC cell lines before and after knockdown of over-amplified transcripts via a high-throughput siRNA–mediated screen. The expression-knockdown of 18 of the 26 genes tested showed a growth suppression ≥30% in at least one cell line (P<0.01). In particular, cell lines derived from late-stage OSCC were more sensitive to the knockdown of G3BP1 than cell lines derived from early-stage OSCC, and the growth suppression was likely caused by increase in apoptosis. Further investigation is warranted to examine the biological role of these genes in OSCC progression and their therapeutic potentials.
Author Summary
Neck lymph node metastasis is the most important prognostic factor in oral squamous cell carcinoma (OSCC). To identify genes associated with this critical step of OSCC progression, we compared DNA copy number aberrations and gene expression differences between tumor cells found in metastatic lymph nodes versus those in non-metastatic primary tumors. We identified 95 transcripts (87 genes) with metastasis-specific genome abnormalities and gene expression. Tested in an independent cohort of 133 OSCC patients, the 95 gene signature was an independent risk factor of disease-specific and overall death, suggesting a disease progression phenotype. We knocked down the expression of over-amplified genes in five OSCC cell lines. Knockdown of 18 of the 26 tested genes suppressed the cell growth in at least one cell line. Interestingly, cell lines derived from late-stage OSCC were more sensitive to the knockdown of G3BP1 than cell lines derived from early-stage OSCC. The knockdown of G3BP1 increased programmed cell death in the p53-mutant but not wild-type OSCC cell lines. Taken together, we demonstrate that CNA–associated transcripts differentially expressed in carcinoma cells with an aggressive phenotype (i.e., metastatic to lymph nodes) can be biomarkers with both prognostic information and functional relevance. Moreover, results suggest that G3BP1 is a potential therapeutic target against late-stage p53-negative OSCC.
doi:10.1371/journal.pgen.1003169
PMCID: PMC3547824  PMID: 23341773
10.  Integrative analysis of DNA copy number and gene expression in metastatic oral squamous cell carcinoma identifies genes associated with poor survival 
Molecular Cancer  2010;9:143.
Background
Lymphotropism in oral squamous cell carcinoma (OSCC) is one of the most important prognostic factors of 5-year survival. In an effort to identify genes that may be responsible for the initiation of OSCC lymphotropism, we examined DNA copy number gains and losses and corresponding gene expression changes from tumor cells in metastatic lymph nodes of patients with OSCC.
Results
We performed integrative analysis of DNA copy number alterations (CNA) and corresponding mRNA expression from OSCC cells isolated from metastatic lymph nodes of 20 patients using Affymetrix 250 K Nsp I SNP and U133 Plus 2.0 arrays, respectively. Overall, genome CNA accounted for expression changes in 31% of the transcripts studied. Genome region 11q13.2-11q13.3 shows the highest correlation between DNA CNA and expression. With a false discovery rate < 1%, 530 transcripts (461 genes) demonstrated a correlation between CNA and expression. Among these, we found two subsets that were significantly associated with OSCC (n = 122) when compared to controls, and with survival (n = 27), as tested using an independent dataset with genome-wide expression profiles for 148 primary OSCC and 45 normal oral mucosa. We fit Cox models to calculate a principal component analysis-derived risk-score for these two gene sets ('122-' or '27-transcript PC'). The models combining the 122- or 27-transcript PC with stage outperformed the model using stage alone in terms of the Area Under the Curve (AUC = 0.82 or 0.86 vs. 0.72, with p = 0.044 or 0.011, respectively).
Conclusions
Genes exhibiting CNA-correlated expression may have biological impact on carcinogenesis and cancer progression in OSCC. Determination of copy number-associated transcripts associated with clinical outcomes in tumor cells with an aggressive phenotype (i.e., cells metastasized to the lymph nodes) can help prioritize candidate transcripts from high-throughput data for further studies.
doi:10.1186/1476-4598-9-143
PMCID: PMC2893102  PMID: 20537188
11.  Stromal myofibroblasts in oral leukoplakia and oral squamous cell carcinoma 
Objectives: Oral leukoplakia (OL) is the main potentially malignant disorder and oral squamous cell carcinoma (OSCC) is the most common malignancy of the oral mucosa. Stromal myofibroblasts play an important role in tumor invasion and metastasis, due to its ability to modify the extracellular matrix. This study aimed to evaluate the presence of stromal myofibroblasts in OL and OSCC. Differences in the presence of myofibroblasts among OL with distinct grades of epithelial dysplasia as well as between histologically high- and low-invasive OSCC were also assessed. Study Design: A total of 30 OL and 41 OSCC from archival formalin-fixed, paraffin-embedded specimens were evaluated. 10 samples of normal oral mucosa were used as a control. Myofibroblasts were identified by immunohistochemical detection of alpha smooth muscle actin and its presence was classified as negative, scanty or abundant. Differences in the presence of myofibroblasts among OL with distinct grades of epithelial dysplasia as well as between high- and low-invasive OSCC were analyzed using the Mann-Whitney test. Results: Myofibroblasts were not detected in normal oral mucosa and OL, whatever its histological grade. In OSCC, the presence of stromal myofibroblasts was classified as negative in 11 (26.8%), scanty in 15 (36.6%), and abundant in 15 samples (36.6%). The presence of stromal myofibroblasts was statistically higher in high-invasive OSCC than in low-invasive OSCC (p<0.05). Conclusions: Stromal myofibroblasts were not detected in OL, indicating that these cells are not important during oral carcinogenesis. Nevertheless, stromal myofibroblasts were heterogeneously detected in OSCC and its presence was higher in tumors with a more diffuse histological pattern of invasion. These findings suggest that myofibroblasts are associated with the creation of a permissive environment for tumor invasion in OSCC.
Key words:Leukoplakia, oral squamous cell carcinoma, myofibroblast.
doi:10.4317/medoral.17834
PMCID: PMC3482514  PMID: 22322518
12.  Expression Analysis of SPARC/Osteonectin in Oral Squamous Cell Carcinoma Patients: From Saliva to Surgical Specimen 
BioMed Research International  2013;2013:736438.
Oral squamous cell carcinoma (OSCC) remains a significant cause of morbidity and mortality, with approximately 540,000 new cases annually worldwide. The molecular mechanisms related to the pathogenesis of this disease are still poorly understood. The discovery of a molecular marker that allows the early detection of this cancer, which can be easily identified in biological samples, such as saliva, without intervening in advanced stages, is a challenge. Numerous studies have identified a panel of molecular markers differently expressed in OSCC and normal oral mucosa. In particular, it was found an aberrant expression of matricellular glycoprotein SPARC. SPARC is involved in normal tissue remodeling, regulating the deposition of extracellular matrix, but also in neoplastic transformation. In fact, aberrant SPARC expression was detected both in stromal cells associated with cancer and in tumor cells. The aim of our study was the evaluation of SPARC on a retrospective series of 119 OSCC cases and the validation of the obtained data on a prospective series of 27 patients with OSCC, of whom we have previously collected saliva, and smeared material. The obtained results were correlated with each other and with clinical pathological parameters at our disposal. The study demonstrated a prognostic value of SPARC, especially with regard to its expression in the stroma surrounding OSCC (P < 0.05).
doi:10.1155/2013/736438
PMCID: PMC3876772  PMID: 24396828
13.  Expression of MAGE-A12 in Oral Squamous Cell Carcinoma 
Disease markers  2007;24(1):27-32.
Melanoma associated-A antigens (MAGE-A) are silent in normal tissues except testis. However, they are activated in a variety of different tumors. Thus, their expression is highly specific to cancer cells. Reverse transcription-nested polymerase chain reaction (RT-nPCR) is a highly sensitive technique that has been used successfully for the detection ofMAGE genes in tissue samples. The aim of the study is to analyze the expression rate of MAGE-A12 in oral squamous cell carcinoma (OSCC) using a high sensitive RT-nPCR. Total of 57 tissue samples obtained from patients with OSCC and 20 normal oral mucosal (NOM) probes of otherwise healthy volunteers were included to this study. No expression of MAGE-A12 was observed in the non-neoplastic NOM tissues. MAGE-A12 was expressed in 49.1% of the investigated tumor samples. The correlation between malignant lesion and MAGE-A12 detection was significant (p < 0.001). It is concluded that results of this study may indicate MAGE-A12 as a useful additional diagnostic marker especially for the early detection of OSCC distinguishing neoplastic transformation and detection of occult and/or rare disseminated cancer cells. In addition, MAGE-A12 expression in OSCC may also determine a new immunotherapeutic target and might be warranted to develop vaccine for OSCC.
doi:10.1155/2008/359840
PMCID: PMC3850551  PMID: 18057533
14.  Tumor and salivary matrix metalloproteinase levels are strong diagnostic markers of oral squamous cell carcinoma 
Background
The matrix metalloproteinases (MMPs) cause degradation of the extracellular matrix and basement membranes, and thus may play a key role in cancer development.
Methods
In our search for biomarkers for oral squamous cell carcinomas (OSCC), we compared primary OSCC, oral dysplasia and control subjects with respect to: (1) expression of MMP1, MMP3, MMP10 and MMP12 in oral epithelial tissue using Affymetrix U133 2.0 Plus GeneChip arrays, followed by qRT-PCR for MMP1, and (2) determination of MMP1 and MMP3 concentrations in saliva.
Results
MMP1 expression in primary OSCC (n=119) was >200-fold higher (p=7.16×10−40) compared with expression levels in non-neoplastic oral epithelium from controls (n=35). qRT-PCR results on 30 cases and 22 controls confirmed this substantial differential expression. The exceptional discriminatory power to separate OSCC from controls was validated in two independent testing sets (AUC%=100; 95% CI, 100-100 and AUC%=98.4; 95% CI, 95.6–100). Salivary concentrations of MMP1 and MMP3 in OSCC patients (33 stage I/II, 26 stage III/IV) were 6.2 times (95% CI, 3.32–11.73) and 14.8 times (95% CI, 6.75–32.56) higher, respectively, than in controls, and displayed an increasing trend with higher stage disease.
Conclusions
Tumor and salivary MMPs are robust diagnostic biomarkers of OSCC.
Impact
The capacity of MMP gene expression to identify OSCC provides support for further investigation into MMPs as potential markers for OSCC development. Detection of MMP proteins in saliva in particular may provide a promising means to detect and monitor OSCC non-invasively.
doi:10.1158/1055-9965.EPI-11-0503
PMCID: PMC3237810  PMID: 21960692
oral squamous cell carcinoma; matrix metalloproteinase; MMP; saliva; gene expression
15.  A gene signature in histologically normal surgical margins is predictive of oral carcinoma recurrence 
BMC Cancer  2011;11:437.
Background
Oral Squamous Cell Carcinoma (OSCC) is a major cause of cancer death worldwide, which is mainly due to recurrence leading to treatment failure and patient death. Histological status of surgical margins is a currently available assessment for recurrence risk in OSCC; however histological status does not predict recurrence, even in patients with histologically negative margins. Therefore, molecular analysis of histologically normal resection margins and the corresponding OSCC may aid in identifying a gene signature predictive of recurrence.
Methods
We used a meta-analysis of 199 samples (OSCCs and normal oral tissues) from five public microarray datasets, in addition to our microarray analysis of 96 OSCCs and histologically normal margins from 24 patients, to train a gene signature for recurrence. Validation was performed by quantitative real-time PCR using 136 samples from an independent cohort of 30 patients.
Results
We identified 138 significantly over-expressed genes (> 2-fold, false discovery rate of 0.01) in OSCC. By penalized likelihood Cox regression, we identified a 4-gene signature with prognostic value for recurrence in our training set. This signature comprised the invasion-related genes MMP1, COL4A1, P4HA2, and THBS2. Over-expression of this 4-gene signature in histologically normal margins was associated with recurrence in our training cohort (p = 0.0003, logrank test) and in our independent validation cohort (p = 0.04, HR = 6.8, logrank test).
Conclusion
Gene expression alterations occur in histologically normal margins in OSCC. Over-expression of the 4-gene signature in histologically normal surgical margins was validated and highly predictive of recurrence in an independent patient cohort. Our findings may be applied to develop a molecular test, which would be clinically useful to help predict which patients are at a higher risk of local recurrence.
doi:10.1186/1471-2407-11-437
PMCID: PMC3198722  PMID: 21989116
oral squamous cell carcinoma; surgical resection margins; global gene expression profiling; prognostic signature; recurrence
16.  A Genetic Expression Profile Associated With Oral Cancer Identifies A Group of Patients at High-Risk of Poor Survival 
Purpose
To determine if gene expression signature of invasive oral squamous cell carcinoma (OSCC) can sub-classify OSCC on the basis of survival.
Experimental Design
We analyzed the expression of 131 genes in 119 OSCC, 35 normal and 17 dysplastic mucosae to identify cluster-defined sub-groups. Multivariate Cox regression was used to estimate the association between gene expression and survival. By stepwise Cox regression the top predictive models of OSCC-specific survival were determined, and compared by Receiver Operating Characteristics (ROC) analysis.
Results
The 3-year overall mean survival (± SE) for a cluster of 45 OSCC patients was 38.7 ± 0.09%, compared to 69.1 ± 0.08% for the remaining patients. Multivariate analysis adjusted for age, sex and stage showed that the 45 OSCC cluster patients had worse overall and OSCC-specific survival (HR=3.31, 95% CI: 1.66, 6.58; HR=5.43, 95% CI: 2.32, 12.73, respectively). Stepwise Cox regression on the 131 probe sets revealed that a model with a term for LAMC2 (laminin, gamma 2) gene expression best identified patients with worst OSCC-specific survival. We fit a Cox model with a term for a principal component analysis-derived risk-score marker (‘PCA’) and two other models that combined stage with either LAMC2 or PCA. The Area Under the Curve for models combining stage with either LAMC2 or PCA was 0.80 or 0.82, respectively, compared to 0.70 for stage alone (p=0.013 and 0.008, respectively).
Conclusions
Gene expression and stage combined predict survival of OSCC patients better than stage alone.
doi:10.1158/1078-0432.CCR-08-1816
PMCID: PMC2683360  PMID: 19228736
17.  Establishment of field change by expression of cytokeratins 8/18, 19, and MMP-9 in an apparently normal oral mucosa adjacent to squamous cell carcinoma: A immunohistochemical study 
Background:
Squamous cell carcinoma is the most frequent malignancy of the oral cavity. The survival rate of this malignancy has not improved from past two decades. The major factors responsible for this could be due to loco regional and distant metastatic spread. However, the other important prognostic factor is concomitant occurrence and recurrence of multiple primary carcinomas in the head and neck region, which is explained as the concept of field cancerization. The evidence to support the field change in normal mucosa of Oral squamous cell carcinoma (OSCC) through biological markers using immunohistochemistry has always been challenging.
Aim:
Hence, the aim of the present research is to identify changes in the expression of CK 8/18, 19, and MMP-9 to visualize field changes in the clinically normal mucosa adjacent to OSCC and compare with non neoplastic normal oral mucosa.
Materials and Methods:
20 cases of OSCC with radical resection specimens were included in the study. Lesional tissue and adjacent normal looking mucosa were taken during grossing. Ten cases of non-neoplastic normal oral mucosa are also included in the study. Markers such as CK 8/18, CK 19, and MMP-9 are used by the immunohistochemical method in this present study.
Result and Conclusion:
The enhance expression of CK 8/18 (80%), CK 19 (70%), and MMP 9 (90%) in ANM was noted and furthermore in six ANM showing severe dysplasia with enhance expression of CK 8/18, CK 19, and MMP 9 in the apparently normal oral mucosa can suggest a field cancerization.
doi:10.4103/0973-029X.92966
PMCID: PMC3303499  PMID: 22438638
Cytokeratin; field cancerization; matrix metalloproteinase
18.  Low prevalence of high risk human papillomavirus in normal oral mucosa by hybrid capture 2 
High risk human papillomavirus (HR-HPV) are recognized as a necessary factor to development cervical cancer. During the last decade many studies have found HR-HPV in oral squamous cell carcinoma (OSCC) and normal oral mucosa, however the association between HR-HPV and OSCC is still uncertain. The aim of the study was to determine DNA HR-HPV in normal oral cavity of healthy adults. A cross-sectional study was performed; samples from 77 patients with normal oral cavity were collected at the Dentistry school, Autonomous University of Yucatan, Merida, Yucatan, México. HR-HPV was detected by hybrid capture 2. One sample out of 77(1.2%) was positive for HR-PVH. It was from a man of 50 years old. HRHPV is present in low rate among healthy oral mucosa. Hybrid capture 2 could be a good methodology for large epidemiology studies.
doi:10.1590/S1517-83822008000100008
PMCID: PMC3768347  PMID: 24031173
Papillomavirus; hybrid capture 2; oral mucosa; squamous cell carcinoma; high risk
19.  Nicotinamide N-Methyltransferase Upregulation Inversely Correlates with Lymph Node Metastasis in Oral Squamous Cell Carcinoma 
Molecular Medicine  2007;13(7-8):415-421.
We investigated expression levels of Nicotinamide N-Methyltransferase (NNMT), an enzyme involved in the biotransformation of many drugs and xenobiotic compounds, in oral squamous cell carcinoma (OSCC). Measurements were performed by semi-quantitative RT-PCR and quantitative real-time PCR in tumor and matched adjacent healthy tissue. Interestingly, NNMT was up-regulated in most of the favorable OSCCs, while no marked NNMT expression alterations between tumor and normal mucosa were detected in most of the unfavorable OSCCs. Western blot and immunohistochemical analyses also were performed and the relationship between tumor characteristics and NNMT levels in OSCC were studied to evaluate the effectiveness of NNMT as a prognostic marker in the squamous cell carcinoma of the oral cavity. In summary, the present study suggests that NNMT may have potential as a biomarker and a therapeutic target for OSCC.
doi:10.2119/2007-00035.Sartini
PMCID: PMC1952675  PMID: 17622326
20.  USP22 Is Useful as a Novel Molecular Marker for Predicting Disease Progression and Patient Prognosis of Oral Squamous Cell Carcinoma 
PLoS ONE  2012;7(8):e42540.
Background and Objective
The significance of ubiquitin-specific protease 22 (USP22) as a potential marker has been growing in the field of oncology. The aim of this study was to investigate the role of USP22 and the association with its potential targets in oral squamous cell carcinoma (OSCC).
Methods
Immunohistochemistry was used to determine the expression of USP22 protein in 319 OSCC patients in comparison with 42 healthy controls. The clinical correlations and prognostic significance of the aberrantly expressed protein was evaluated to identify novel biomarker of OSCC.
Results
The incidence of positive USP22 expression was 63.32% in 319 conventional OSCC tissues. The protein expression level of USP22 was concomitantly up-regulated from non-cancerous mucosa to primary carcinoma and from carcinomas to lymph node metastasis (P<0.001). Moreover, statistical analysis showed that positive USP22 expression was positively related to lymph node metastasis, Ki67, Cox-2 and recurrence. Furthermore, it was shown that patients with positive USP22 expression had significantly poorer outcome compared with patients with negative expression of USP22 for patients with positive lymph nodes. Multivariate Cox regression analysis revealed that USP22 expression level was an independent prognostic factor for both overall survival and disease-free survival (P<0.001 and P<0.001, respectively). Cancer cells with reduced USP22 expression exhibited reduced proliferation and colony formation evaluated by MTT and soft agar assays.
Conclusion
To our knowledge, this is the first study that determines the relationship between USP22 expression and prognosis in OSCC. We found that increased expression of USP22 is associated with poor prognosis in OSCC. USP22 may represent a novel and useful prognostic marker for OSCC.
doi:10.1371/journal.pone.0042540
PMCID: PMC3411815  PMID: 22880026
21.  Programmed cell death 4 loss increases tumor cell invasion and is regulated by miR-21 in oral squamous cell carcinoma 
Molecular Cancer  2010;9:238.
Background
The tumor suppressor Programmed Cell Death 4 (PDCD4) has been found to be under-expressed in several cancers and associated with disease progression and metastasis. There are no current studies characterizing PDCD4 expression and its clinical relevance in Oral Squamous Cell Carcinoma (OSCC). Since nodal metastasis is a major prognostic factor in OSCC, we focused on determining whether PDCD4 under-expression was associated with patient nodal status and had functional relevance in OSCC invasion. We also examined PDCD4 regulation by microRNA 21 (miR-21) in OSCC.
Results
PDCD4 mRNA expression levels were assessed in 50 OSCCs and 25 normal oral tissues. PDCD4 was under-expressed in 43/50 (86%) OSCCs, with significantly reduced mRNA levels in patients with nodal metastasis (p = 0.0027), and marginally associated with T3-T4 tumor stage (p = 0.054). PDCD4 protein expression was assessed, by immunohistochemistry (IHC), in 28/50 OSCCs and adjacent normal tissues; PDCD4 protein was absent/under-expressed in 25/28 (89%) OSCCs, and marginally associated with nodal metastasis (p = 0.059). A matrigel invasion assay showed that PDCD4 expression suppressed invasion, and siRNA-mediated PDCD4 loss was associated with increased invasive potential of oral carcinoma cells. Furthermore, we showed that miR-21 levels were increased in PDCD4-negative tumors, and that PDCD4 expression may be down-regulated in OSCC by direct binding of miR-21 to the 3'UTR PDCD4 mRNA.
Conclusions
Our data show an association between the loss of PDCD4 expression, tumorigenesis and invasion in OSCC, and also identify a mechanism of PDCD4 down-regulation by microRNA-21 in oral carcinoma. PDCD4 association with nodal metastasis and invasion suggests that PDCD4 may be a clinically relevant biomarker with prognostic value in OSCC.
doi:10.1186/1476-4598-9-238
PMCID: PMC2949797  PMID: 20831814
22.  Gα12 Drives Invasion of Oral Squamous Cell Carcinoma through Up-Regulation of Proinflammatory Cytokines 
PLoS ONE  2013;8(6):e66133.
Oral squamous cell carcinoma (OSCC) ranks among the top ten most prevalent cancers worldwide. Like most head and neck squamous cell carcinomas (HNSCCs), OSCC is highly inflammatory and aggressive. However, the signaling pathways triggering the activation of its inflammatory processes remain elusive. G protein-coupled receptor signaling regulates the inflammatory response and invasiveness of cancers, but it remains unclear whether Gα12 is a critical player in the inflammatory cytokine pathway during the tumorigenesis of OSCC. This study was undertaken to determine the role of Gα12 signaling in the regulation of proinflammatory cytokines in their mediation of OSCC invasion. We found that both the transcription and protein levels of Gα12 are up-regulated in OSCC tumors. The elevated Gα12 expressions in OSCC patients also correlated with extra-capsular spread, an indicator of tumor invasiveness in HNSCCs. This clinical finding was supported by the studies of overexpression and RNAi knockdown of Gα12 in OSCC cells, which demonstrated that Gα12 promoted tumor cell migration and invasion. To understand how Gα12 modulates OSCC invasiveness, we analyzed key biological processes in microarray data upon depletion of Gα12 and found that cytokine- and other immune-related pathways were severely impaired. Importantly, the mRNA levels of IL-6 and IL-8 proinflammatory cytokines in clinical samples were found to be significantly correlated with the increased Gα12 levels, suggesting a potential role of Gα12 in modulating the IL-6 and IL-8 expressions. Supporting this hypothesis, overexpression or RNAi knockdown of Gα12 in OSCC cell lines both showed that Gα12 positively regulated the mRNA and protein levels of IL-6 and IL-8. Finally, we demonstrated that the Gα12 promotion of tumor cell invasiveness was suppressed by the neutralization of IL-6 and IL-8 in OSCC cells. Together, these findings suggest that Gα12 drives OSCC invasion through the up-regulation of IL-6 and IL-8 cytokines.
doi:10.1371/journal.pone.0066133
PMCID: PMC3676329  PMID: 23762476
23.  Homeobox gene expression profile indicates HOXA5 as a candidate prognostic marker in oral squamous cell carcinoma 
International Journal of Oncology  2011;40(4):1180-1188.
The search for molecular markers to improve diagnosis, individualize treatment and predict behavior of tumors has been the focus of several studies. This study aimed to analyze homeobox gene expression profile in oral squamous cell carcinoma (OSCC) as well as to investigate whether some of these genes are relevant molecular markers of prognosis and/or tumor aggressiveness. Homeobox gene expression levels were assessed by microarrays and qRT-PCR in OSCC tissues and adjacent non-cancerous matched tissues (margin), as well as in OSCC cell lines. Analysis of microarray data revealed the expression of 147 homeobox genes, including one set of six at least 2-fold up-regulated, and another set of 34 at least 2-fold down-regulated homeobox genes in OSCC. After qRT-PCR assays, the three most up-regulated homeobox genes (HOXA5, HOXD10 and HOXD11) revealed higher and statistically significant expression levels in OSCC samples when compared to margins. Patients presenting lower expression of HOXA5 had poorer prognosis compared to those with higher expression (P=0.03). Additionally, the status of HOXA5, HOXD10 and HOXD11 expression levels in OSCC cell lines also showed a significant up-regulation when compared to normal oral keratinocytes. Results confirm the presence of three significantly upregulated (>4-fold) homeobox genes (HOXA5, HOXD10 and HOXD11) in OSCC that may play a significant role in the pathogenesis of these tumors. Moreover, since lower levels of HOXA5 predict poor prognosis, this gene may be a novel candidate for development of therapeutic strategies in OSCC.
doi:10.3892/ijo.2011.1321
PMCID: PMC3584618  PMID: 22227861
oral cancer; homeobox genes; HOXA5; prognosis
24.  The expression profile of Oct4 and Sox2 in the carcinogenesis of oral mucosa 
This study is to detect the co-expression of embryonic stem cell-related markers (Oct4 and Sox2) in the carcinogenesis of oral mucosa. The expression profile of these markers was studied by immunohistochemistry assay in rat and human samples. The normal oral mucosa (20 cases) and the transforming oral mucosa (20 cases) were performed in rat samples. The precancerous lesions (20 cases), OSCCs in primary site (116 cases), corresponding epithelial non-cancer tissues adjacent to the OSCC (20 cases) and 46 paired metastatic OSCCs in lymph nodes were performed in human samples. The co-expression of the two markers was defined as both of them are positively detected in the same site of one case under one selected field of microscope. The results indicated that Oct4 and Sox2 were individually detected in normal oral mucosa, but they cannot be co-expressed in the same site of one case. The co-expression of Oct4 and Sox2 (Oct4+Sox2+) was frequently detected in the transforming oral mucosa of rat (16/20), precancerous lesions of human (12/20) and epithelial non-cancer tissues adjacent to the OSCC (18/20). Also, Oct4+Sox2+ profile was remarkable noted in the primary sites of OSCCs (38/116). In the 46 paired OSCCs (primary sites with lymph node metastasis), Oct4+Sox2+ profile (8/46) was less frequently detected than Oct4low/-Sox2low/- (14/46) profile in the metastatic sites. To conclude, this study suggests Oct4 and Sox2 are expressed in normal oral mucosa, premalignant diseases, primary sites of OSCCs and metastasis sites of OSCCs. Oct4+Sox2+ profile may contribute to the malignant transformation of oral mucosa.
PMCID: PMC3885457  PMID: 24427323
Carcinogenesis; Oct4; Sox2
25.  MicroRNA Alterations and Associated Aberrant DNA Methylation Patterns across Multiple Sample Types in Oral Squamous Cell Carcinoma 
PLoS ONE  2011;6(11):e27840.
Background
MicroRNA (miRNA) expression is broadly altered in cancer, but few studies have investigated miRNA deregulation in oral squamous cell carcinoma (OSCC). Epigenetic mechanisms are involved in the regulation of >30 miRNA genes in a range of tissues, and we aimed to investigate this further in OSCC.
Methods
TaqMan® qRT-PCR arrays and individual assays were used to profile miRNA expression in a panel of 25 tumors with matched adjacent tissues from patients with OSCC, and 8 control paired oral stroma and epithelium from healthy volunteers. Associated DNA methylation changes of candidate epigenetically deregulated miRNA genes were measured in the same samples using the MassArray® mass spectrometry platform. MiRNA expression and DNA methylation changes were also investigated in FACS sorted CD44high oral cancer stem cells from primary tumor samples (CSCs), and in oral rinse and saliva from 15 OSCC patients and 7 healthy volunteers.
Results
MiRNA expression patterns were consistent in healthy oral epithelium and stroma, but broadly altered in both tumor and adjacent tissue from OSCC patients. MiR-375 is repressed and miR-127 activated in OSCC, and we confirm previous reports of miR-137 hypermethylation in oral cancer. The miR-200 s/miR-205 were epigenetically activated in tumors vs normal tissues, but repressed in the absence of DNA hypermethylation specifically in CD44high oral CSCs. Aberrant miR-375 and miR-200a expression and miR-200c-141 methylation could be detected in and distinguish OSCC patient oral rinse and saliva from healthy volunteers, suggesting a potential clinical application for OSCC specific miRNA signatures in oral fluids.
Conclusions
MiRNA expression and DNA methylation changes are a common event in OSCC, and we suggest miR-375, miR-127, miR-137, the miR-200 family and miR-205 as promising candidates for future investigations. Although overall activated in OSCC, miR-200/miR-205 suppression in oral CSCs indicate that cell specific silencing of these miRNAs may drive tumor expansion and progression.
doi:10.1371/journal.pone.0027840
PMCID: PMC3222641  PMID: 22132151

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