Progression Of Oral Leukoplakia Research Articles

CircHLA-C: A significantly upregulated circRNA co-existing in oral leukoplakia and oral lichen planus

ABSTRACT Background Oral leukoplakia (OLK) and oral lichen planus (OLP) are common precancerous lesions of the oral mucosa. The role of circular RNAs (circRNAs) in OLK and OLP is unclear. The aim of this study was to evaluate the circRNA expression profiles of OLK and OLP, and further explore the potential role of circRNAs in the pathogenesis of these two diseases. Methods High throughput sequencing technology was performed to detect the differentially expressed circRNA in OLK (n = 6), OLP (n = 6), oral squamous cell carcinoma (n = 6), and normal oral mucosa tissues (n = 6). Expression of selected circRNAs was validated by qRT-PCR, enzyme tolerance assay, and Sanger sequencing. Expanded sample size validation was done in 20 tissue pairs. The biological processes and signal pathways involved in differential circRNA were analyzed by GO and KEGG enrichment. TargetScan and MiRanda were used to predict miRNAs downstream of circRNA and draw competitive endogenous RNA network diagram. Results Forty-nine circRNAs were significantly altered in OLK and OLP, including 30 upregulated and 19 downregulated circRNAs. The five selected circRNAs were validated by qRT-PCR, Sanger sequencing, and RNase R assay. GO and KEGG analyses indicated that the upregulated circHLA-C may be involved in the biological process of immune function of OLK and OLP. Bioinformatics analysis indicated that circHLA-C may be involved in the progression of OLK and OLP as a ceRNA. In validation with expanded sample size, PCR results showed that circHLA-C expression was significantly upregulated in OLK and OLP. ROC analysis indicated that circHLA-C has potential diagnostic value with good accuracy and specificity. Conclusion Our study revealed that circHLA-C is the most significantly upregulated circRNA co-existing in OLK and OLP, and we preliminarily discuss the role of circHLA-C in the etiopathogenesis and progression of OLK and OLP.

A novel lncRNA LOLA1 may predict malignant progression and promote migration, invasion, and EMT of oral leukoplakia via the AKT/GSK-3β pathway.

Although dysregulation and dysfunction of long noncoding RNAs (lncRNAs) have been implicated in malignant behavior of oral squamous cell carcinoma (OSCC), whether aberrant lncRNAs play a role in the carcinogenesis of oral leukoplakia (OL) as the best-known precursor of OSCC remains undetermined. Differentially expressed lncRNAs in the occurrence and progression of OL were studied by microarray and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). We found a novel key lncRNA n386251 that we named LOLA1 (lncRNA oral leukoplakia progressed associated 1) in the OL progression. The results of qRT-PCR revealed that LOLA1 aberrant expression was validated in tissue samples and cell lines from the normal oral mucosa, OL to OSCC. Fluorescent in situ hybridization showed that LOLA1 expression localized predominately at the cytoplasm of Leuk1 cells. Cell function assays showed that LOLA1 significantly influenced cell migration, invasion, and epithelial-mesenchymal transition (EMT) protein expression. Potential mechanism experiments revealed that AKT/GSK-3β signaling was involved in the regulatory mechanism of LOLA1 in OL progression. Remarkably, Kaplan-Meier analysis revealed that LOLA1 overexpression could predict malignant events of OL progression to OSCC. In conclusion, the current study for the first time profiled and validated the key lncRNAs related to OL progression. Importantly, we demonstrated that a novel lncRNA LOLA1 upregulation was associated with OL malignant progression, suggesting LOLA1 may be a predictive biomarker. Moreover, LOLA1 may promote migration, invasion, and EMT process in OL malignant progression via AKT/GSK-3β pathway.

Importance of the PD-1/PD-L1 Axis for Malignant Transformation and Risk Assessment of Oral Leukoplakia.

Background: The programmed cell death ligand 1/programmed cell death receptor 1 (PD-L1/PD-1) Immune Checkpoint is an important modulator of the immune response. Overexpression of the receptor and its ligands is involved in immunosuppression and the failure of an immune response against tumor cells. PD-1/PD-L1 overexpression in oral squamous cell carcinoma (OSCC) compared to healthy oral mucosa (NOM) has already been demonstrated. However, little is known about its expression in oral precancerous lesions like oral leukoplakia (OLP). The aim of the study was to investigate whether an increased expression of PD-1/PD-L1 already exists in OLP and whether it is associated with malignant transformation. Material and Methods: PD-1 and PD-L1 expression was immunohistologically analyzed separately in the epithelium (E) and the subepithelium (S) of OLP that had undergone malignant transformation within 5 years (T-OLP), in OLP without malignant transformation (N-OLP), in corresponding OSCC and in NOM. Additionally, RT-qPCR analysis for PD-L1 expression was done in the entire tissues. Additionally, the association between overexpression and malignant transformation, dysplasia and inflammation were examined. Results: Compared to N-OLP, there were increased levels of PD-1 protein in the epithelial and subepithelial layers of T-OLP (pE = 0.001; pS = 0.005). There was no significant difference in PD-L1 mRNA expression between T-OLP and N-OLP (p = 0.128), but the fold-change increase between these groups was significant (Relative Quantification (RQ) = 3.1). In contrast to N-OLP, the PD-L1 protein levels were significantly increased in the epithelial layers of T-OLP (p = 0.007), but not in its subepithelial layers (p = 0.25). Importantly, increased PD-L1 levels were significantly associated to malignant transformation within 5 years. Conclusion: Increased levels of PD-1 and PD-L1 are related to malignant transformation in OLP and may represent a promising prognostic indicator to determine the risk of malignant progression of OLP. Increased PD-L1 levels might establish an immunosuppressive microenvironment, which could favor immune escape and thereby contribute to malignant transformation. Hence, checkpoint inhibitors could counteract tumor development in OLP and may serve as efficient therapeutic strategy in patients with high-risk precancerous lesions.

Transcriptome array screening and verification of oral leukoplakia carcinogenesis-related hypoxia-responsive gene and microRNA.

To study the hypoxia response gene and microRNA (miRNA) expression profiles in the pathogenesis and progression of oral leukoplakia (OLK). Affymetrix GeneChip human transcriptome array 2.0 was used to detect the transcriptome of normal mucosa, low-risk OLK, high-risk OLK, and early squamous cell carcinoma (SCC). Gene ontology function analysis was used to screen genes and key miRNAs whose biological role is hypoxia response. Quantitative reverse transcription polymerase ch-ain reaction (qRT-PCR) was used to verify the expression of hypoxia response genes and miRNAs. A total of 7 different genes of hypoxia response between normal mucosa and low-risk OLK, 10 genes between low-risk and high-risk OLK, and 21 genes between high-risk OLK and SCC were identified. The results of qRT-PCR showed that the expression of hypoxia-inducible factor 1α, chemokine cc-motif ligand 2, and matrix metalloproteinase 3 mRNA and miR-21 in normal mucosa, OLK, and SCC increased in a stepwise manner. The expression difference between OLK and SCC was statistically significant and consistent with the results of transcriptome array. The hypoxia response gene and related miRNA play roles in the development and progression of OLK.

Correlation of vascularization and inflammation with severity of oral leukoplakia

Changes in submucosal vascularization and inflammation, determined by immunohistochemistry staining, were shown to be correlated with the development of dysplasia and invasiveness of epithelial cells in premalignant and malignant lesions. This study evaluated changes in sections routinely stained with Hematoxylin and Eosin (H&E) in order to investigate vascular density and intensity of inflammatory cells infiltration during the progression of oral leukoplakia with mild dysplasia to Squamous Cell Carcinoma (SCC).The aim of the research was to determine whether changes in sub-mucosal vascularity and inflammatory infiltration of leukoplakia in routine H&E-stained sections could contribute to the assessment of severity of the lesion. In this cross-sectional, comparative and descriptive study, vascular density and inflammation intensity of 125 available samples of H&E-stained sections, consisting of 35 cases of mild and moderate dysplasia, 38 severe dysplasia and carcinoma in situ, and 52 SCC, were investigated. To analyze the data, chi-square test, Mann-Whitney test, Kruskal-Wallis test, Tukey's post hoc test, and cumulative ordinal logistic regression were conducted. There was a significantly higher vascular density in cases with severe dysplasia, in situ carcinoma, and SCC compared to those with mild to moderate dysplasia (P<0.0001). However, the difference in vascularity was not statically significant between severe dysplasia, carcinoma in situ, and SCC (P=0.78). Intensity of inflammatory cells infiltration in the underlying connective tissue was significantly different among the three groups (P<0.0001), and the highest intensity of inflammatory cells infiltration was seen in the SCC group. Increased submucosal vascularization and inflammatory cells infiltration can contribute further to predicting more aggressive epithelial dysplasia.

Signal regulatory protein α associated with the progression of oral leukoplakia and oral squamous cell carcinoma regulates phenotype switch of macrophages.

Signal regulatory protein α (SIRPα) is a cell-surface protein expressed on macrophages that are regarded as an important component of the tumor microenvironment. The expression of SIRPα in oral leukoplakia (OLK) and oral squamous cell carcinoma (OSCC), and further explored the role of SIRPα on the phenotype, phagocytosis ability, migration, and invasion of macrophages in OSCC were investigated. The expression of SIRPα in OLK was higher than in OSCC, correlating with the expression of CD68 and CD163 on macrophages. After cultured with the conditioned media of oral cancer cells, the expression of SIRPα on THP-1 cells was decreased gradually. In co-culture system, macrophages were induced into M2 phenotype by oral cancer cells. Blockade of SIRPα inhibited phagocytosis ability and IL-6, TNF-α productions of macrophages. In addition, the proliferation, migration, and IL-10, TGF-β productions of macrophages were upregulated after blockade of SIRPα. Macrophages upregulated the expression of SIRPα and phagocytosis ability, and inhibited the migration and invasion when the activation of NF-κB was inhibited by pyrrolidine dithiocarbamate ammonium (PDTC). Hence, SIRPα might play an important role in the progression of OLK and oral cancer, and could be a pivotal therapeutic target in OSCC by regulating the phenotype of macrophages via targeting NF-κB.

MicroRNA expression profiling and functional annotation analysis of their targets associated with the malignant transformation of oral leukoplakia

In spite the tremendous achievements that have been acquired in the field of molecular biology, the underlying mechanism associated with malignant transformed oral leukoplakia (OLK) is still unclear and poorly understood. The aim of this study is to investigate the microRNA (miRNA) expression profiles in OLK and its aggressive transformed tissues from the white lesion of human oral mucosa. The original miRNA expression dataset was downloaded from Gene Expression Omnibus (GEO) database and differentially expressed miRNAs were identified using two-sample t test method. Unsupervised hierarchical clustering and principal component analysis of these differentially expressed miRNAs indicated that 38-miRNA candidates could significantly discriminate OLK from malignant transformed oral mucosa samples. Besides, potential transcription factors were predicted using CyTargetLinker plugin and the miRNA–mRNA regulatory network associated with the malignant pathogenesis was visualized in Cytoscape environment. Totally, 3-miRNA signatures (miR-129-5p, miR-339-5p and miR-31*) were found to be hubs that mediated the initiation and progression of OLK from the non-malignant to the aggressive one via targeting various transcription factors. Functional enrichment analysis based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) suggested that the dysregulation of immune response was responsible for oral carcinogenesis. In conclusion, we constructed a miRNA–mRNA regulatory network associated with the malignant transformation of OLK, and screened out some miRNAs and transcription factors that may have prominent roles during OLK malignant progression.

Influence of thiozolidinediones (TZD) exposure on the progression of oral leukoplakia (L) and erythroplakia (E) to the development of head and neck cancer (HNC).

1565 Background: Oral L&E are precancerous conditions. TZDs, PPARγ ligands used to treat type II diabetes mellitus (DM) have been shown to have in vitro antineoplastic activity. Statistically significant reduction in the HNC cancer has been reported on exposure to TZD. TZD have been evaluated in clinical trials to treat leukoplakia. We conducted a retrospective cohort analysis to evaluate the efficacy of TZDs in preventing the development of HNC in subjects with oral L&E who are exposed to TZDs. Methods: L, E HNC, and DM were identified using ICD 9 codes from the VISN 16 patient data base serving 10 VA health care facilities from to 1996 to 2008. Anti-DM drug history was obtained from pharmacy data. The association between TZD exposure and the development of HNC was evaluated using multivariate Cox regression with age and tobacco use as baseline covariates, anti-DM drug exposures as time-dependent covariates, with stratification on race. Results: 2,363 subjects were identified as having L or E. 115 subjects developed HNC during a total follow-up of 10283 years. Median age, yrs. (quartiles) 59 (52–67); males 2,264 (96%); females 99 (4%); Caucasians 1,647 (70%), African American 450 (19%); others/unknown race 266 (11%). Tobacco users 1,238 (52%); DM subjects 732 (31%). HNC rate was 0.8% per year in TZD users versus 1.1%/year in nonusers (HR 0.79; p=0.4) (Table). Conclusions: TZD exposure did not influence the progression of L&E to HNC. TZD use may decrease the incidence of HNC with out preexisting L&E. Exposure summary and multivariate Cox-regression analysis results Treatment Exposure summary Adjusted HR3(95% confidence limits) p value N (%1) Years (%2) HNC No drug 1,784 (75%) 8,013 (78%) 100 1.00 (reference) - TZDs 131 (5%) 455 (4%) 4 0.81 (0.18–3.71) 0.79 Insulin 228 (9%) 751 (7%) 6 1.00 (0.41–2.41) 0.99 Metformin 394 (16%) 1,416 (14%) 10 0.60 (0.25–1.46) 0.26 Sulfonylureas 392 (16%) 1,624 (16%) 12 0.84 (0.41–1.69) 0.62 1 % of 2,363 subjects. 2 % of 10,283 years of follow-up. 3 Hazard ratio of each treatment adjusting for the presence of age, tobacco, and the other treatments. No significant financial relationships to disclose.

Evaluation of cornulin, keratin 4, keratin 13 expression and grade of dysplasia for predicting malignant progression of oral leukoplakia

Oral leukoplakia is defined as a white patch in the oral cavity that cannot be diagnosed as any other known disorder. These lesions carry an increased risk of malignant progression, and approximately 2-3% per year do progress to cancer. At present biopsies are histopathologically graded for dysplasia to assess the risk of progression, but this grading is somewhat subjective and of limited use for the individual patient. In a previous study we discovered by a comprehensive proteomics approach that compared to normal mucosa, protein expression of cornulin, keratin 4 and keratin 13 is decreased in tumors and severe dysplasia, preneoplastic tissue with a high risk of malignant progression. Here, we studied whether loss of expression of these proteins can predict malignant transformation of oral leukoplakia. Biopsies of 12 progressing and 36 non-progressing leukoplakia lesions were analyzed for cornulin, keratin 4 and keratin 13 expression by immunohistochemistry, and graded for dysplasia. Kaplan-Meier analysis showed that loss of expression of neither cornulin (p=0.075), keratin 4 (p=0.789) nor keratin 13 (p=0.732) was significantly associated with malignant transformation of leukoplakia lesions. However, decreased expression of these proteins was significantly associated with the presence of hyperkeratosis. Only dysplasia grading correlated significantly with malignant progression of leukoplakia (p=0.024). Despite the promising outlook that decreased cornulin, keratin 4 and keratin 13 expression in the oral mucosa is associated with a premalignant state, these markers do not predict malignant transformation of leukoplakia lesions. The most likely explanation is that the aberrant differentiation state of hyperkeratotic leukoplakia lesions already causes a decreased expression, obscuring the putative association with malignant transformation. Our results support the significance of dysplasia grading for the prediction of malignant transformation.

Mast Cells and Angiogenesis in Oral Malignant and Premalignant Lesions

Mast cell contribution to neoangiogenesis during tumorigenesis in oral squamous cell carcinoma is not determined yet. Objectives: To associate numerical mast cell density (MCD) to numerical microvessel density (MVD) during the progression of oral leukoplakia without dysplasia and leukoplakia with dysplasia to squamous cell carcinoma (OSCC). Materials and methods: MVD was analysed immunohistochemically (mouse monoclonal anti-human CD34) in 49 paraffin-embedded specimens, 35 OSCCs, 9 leukoplakias and 5 normal oral tissues. Toluidine blue counterstaining revealed mast cells. MCD and MVD were assessed at the same optical field. Results: MVD increased between: normal oral mucosa, dysplasia (p=0.004), OSCC (p=0.001), leukoplakia and OSCC (p=0.041). MCD increased between: normal oral mucosa, dysplasia (p=0.003), OSCC (p=0.000), leukoplakia and OSCC (p=0.007). MVD was found to depend on MCD (p=0.000) in a percent 28.3% (power curve fit model). Conclusions: Mast cells are attracted at the lesion site and may turn on an angiogenic switch during tumorigenesis in OSCC.

Gene expression profiles of oral leukoplakia and carcinoma: Genome-wide comparison analysis using oligonucleotide microarray technology

Although leukoplakia is the most common precancerous lesion of the oral cavity, its molecular biological properties are largely unknown. The aim of this study was to identify the genes responsible for its pathogenesis and malignant transformation using oligonucleotide microarray technology. The expression profiles of 8,800 genes in human oral leukoplakia (n=4) and oral squamous cell carcinoma (OSCC) (n=2) were analyzed using the Affymetrix GeneChip system and the results were confirmed with RT-PCR. Eight genes were up-regulated (>2.0-fold) and ten were down-regulated (<0.5-fold) in all leukoplakias analyzed with the GeneChip. In particular, loricrin and keratins displayed greater differences between normal tissue and leukoplakia. Some of the 18 alternatively expressed genes were markedly down-regulated in squamous cell carcinoma compared with leukoplakia. Our data suggested that gene abnormalities in cytoskeleton network components might be responsible for the development and progression of oral leukoplakia.