Differential expression of perlecan receptors, α‐dystroglycan and integrin β1, before and after invasion of oral squamous cell carcinoma

J Oral Pathol Med (2011) 40 : 552–559 Objectives: The deposition of perlecan, a heparan sulfate proteoglycan, is enhanced within oral carcinoma in situ (CIS) foci, while it dynamically switches from CIS foci to the stromal space in squamous cell carcinoma (SCC). Because α‐dystroglycan and integrin β1 have been identified as two of the perlecan receptors, we wanted to determine their differential distributions before and after invasion of oral SCC. Methods: Eighty‐two surgical tissue specimens of oral SCC containing different precancerous stages were examined by immunohistochemistry for perlecan, α‐dystroglycan, integrin β1, and Ki‐67. In addition, α‐dystroglycan mRNA signals were localized by in situ hybridization. Results: In normal epithelia, α‐dystroglycan and integrin β1 were localized on the cell membrane of basal cells, while perlecan was faintly present in the intercellular spaces of parabasal cells. In epithelial dysplasia and CIS, α‐dystroglycan and perlecan were well co‐localized in the epithelial layer, especially in its lower half, and this co‐localization was mostly overlapped with Ki‐67‐positive (+) cell zones. However, in SCC, α‐dystroglycan was localized neither within carcinoma cell nests nor in the stroma, while perlecan disappeared from SCC foci but emerged in the stromal space, leaving integrin β1+ and Ki‐67+ cells only to the periphery of SCC foci. α‐Dystroglycan mRNA signals were basically identical to the α‐dystroglycan protein localizations. Conclusion: The findings suggest that α‐dystroglycan and integrin β1 act as perlecan receptors in oral precancerous lesions prior to invasion, and that the perlecan signals via the two different receptors function in cellular differentiation and proliferation of CIS cells, respectively.     

Hypoxia inducible factor‐1α expression in areca quid chewing‐associated oral squamous cell carcinomas

Oral Diseases (2010) 16 , 696–701 Objectives: Hypoxia inducible factor (HIF)‐1α gene expression is mainly induced by tissue hypoxia. Overexpression of HIF‐1α has been demonstrated in a variety of cancers. The aim of this study was to compare HIF‐1α expression in normal human oral epithelium and areca quid chewing‐associated oral squamous cell carcinoma (OSCC) and further to explore the potential mechanisms that may lead to induce HIF‐1α expression. Methods: Twenty‐five OSCC from areca quid chewing‐associated OSCC and 10 normal oral tissue biopsy samples without areca quid chewing were analyzed by immunohistochemistry. The oral epithelial cell line GNM cells were challenged with arecoline, a major areca nut alkaloid, by using Western blot analysis. Furthermore, glutathione precursor N‐acetyl‐l ‐cysteine (NAC), AP‐1 inhibitor curcumin, extracellular signal‐regulated protein kinase inhibitor PD98059, and protein kinase C inhibitor staurosporine were added to find the possible regulatory mechanisms. Results: Hypoxia inducible factor‐1α expression was significantly higher in OSCC specimens than normal specimen (P < 0.05). Arecoline was found to elevate HIF‐1α expression in a dose‐ and time‐dependent manner (P < 0.05). The addition of NAC, curcumin, PD98059, and staurosporine markedly inhibited the arecoline‐induced HIF‐1α expression (P < 0.05). Conclusions: Hypoxia inducible factor‐1α expression is significantly upregulated in areca quid chewing‐associated OSCC and HIF‐1α expression induced by arecoline is downregulated by NAC, curcumin, PD98059, and staurosporine.     

Co‐expression of Hif1α and CAIX is associated with poor prognosis in oral squamous cell carcinoma patients

J Oral Pathol Med (2010) 39 : 313–317 Background: This study investigates the prognostic impact of the expression of hypoxia‐inducible factor 1α (Hif1α) and carbonic anhydrase IX (CAIX) detected by immunohistochemistry in oral squamous cell carcinoma (OSCC). Methods: Statistical analysis of immunohistochemical results with clinical parameters including survival outcomes was performed for 80 OSCC patients. Results: Patients with a low expression of both proteins survived on average 54.8 months, whereas those with an increased expression of Hif1α in their tumors combined with a low expression of CAIX survived on average only 37.6 months (P = 0.026). In multivariate Cox’s regression hazard analysis, again patients with a low expression of Hif1α/CAIX had the best prognosis, whereas patients with increased Hif1α and low CAIX expression carried a 4.97‐fold increased risk of tumor‐related death (P = 0.042). Conclusion: A co‐detection of low Hif1α/CAIX expression is significantly correlated with a better prognosis for OSCC patients, which may have implications for therapy options for these patients.     

The role of cyclin‐dependent kinases in oral potentially malignant disorders and oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is a major global health problem with a relatively low‐moderate 5‐year survival rate. OSCC is often preceded by lesions and conditions known as oral potentially malignant disorders (OPMDs) that have an increased risk of malignant transformation. Despite advances in diagnostic technology and cancer research, the prognosis of OSCC remains poor as it is frequently detected a late stage. Understanding the molecular pathways involved in oral carcinogenesis provides a platform to identify biomarkers that may allow the early detection of OSCC and accurate prediction of the malignant potential of OPMDs. In addition, specific molecular inhibitors can be developed to target these important pathways and allow advanced therapeutic management to improve the prognosis of this malignancy. A common feature across a number of different cancers is the dysfunction of cell cycle moderator proteins known as cyclin‐dependent kinases. This review summarises the current literature regarding the role of cyclin‐dependent kinases in oral carcinogenesis with a particular focus on cyclin‐dependent kinases 4 (CDK4) and 6 (CDK6). This is of particular relevance as CDK4 and CDK6 inhibitors have shown some promising results in other cancer types and are interesting potential treatments for OSCC.     

Histone modifications in oral squamous cell carcinoma and oral potentially malignant disorders

Oral squamous cell carcinoma (OSCC) is a common malignancy with dismal prognosis without effective therapeutic options in advanced cases. The evolution from oral potentially malignant disorders to OSCC has poorly described underlying epigenetic features. With the ability of silencing or activation of vital genes, histone modifications’ and modifiers’ potentiality for early diagnosis, prognosis predicting, and therapy in OSCC were evaluated by extensive epigenetic studies. This review investigates the roles of dysregulated histone modifications and the associated modifying enzymes in OSCC onset and progression. Also, we focus on the current advances of histone modifications as therapeutic targets and the potential value of epi‐drugs.     

Programmable bio‐nanochip‐based cytologic testing of oral potentially malignant disorders in Fanconi anemia

Fanconi anemia (FA) is caused by mutations of DNA repair genes. The risk of oral squamous cell carcinoma (OSCC) among FA patients is 800‐folds higher than in the general population. Early detection of OSCC, preferably at it precursor stage, is critical in FA patients to improve their survival. In an ongoing clinical trial, we are evaluating the effectiveness of the programmable bio‐nanochip (p‐BNC)‐based oral cytology test in diagnosing oral potentially malignant disorders (OPMD) in non‐FA patients. We used this test to compare cytomorphometric and molecular biomarkers in OSCC cell lines derived from FA and non‐FA patients to brush biopsy samples of a FA patient with OPMD and normal mucosa of healthy volunteers. Our data showed that expression patterns of molecular biomarkers were not notably different between sporadic and FA‐OSCC cell lines. The p‐BNC assay revealed significant differences in cytometric parameters and biomarker MCM2 expression between cytobrush samples of the FA patient and cytobrush samples of normal oral mucosa obtained from healthy volunteers. Microscopic examination of the FA patient's OPMD confirmed the presence of dysplasia. Our pilot data suggests that the p‐BNC brush biopsy test recognized dysplastic oral epithelial cells in a brush biopsy sample of a FA patient.     

Transforming growth factor‐β‐regulated fractalkine as a marker of erosive bone invasion in oral squamous cell carcinoma

Patients with oral squamous cell carcinoma (OSCC) bone invasion are surgically treated with bone resection, which results in severe physical and psychological damage. Here, we investigated the potential of fractalkine (CX3CL1), which is regulated by transforming growth factor (TGF‐β), as a novel biomarker for correct prediction and early detection of OSCC‐associated bone invasion. TGF‐β knockdown and treatment with a TGF‐β‐neutralizing antibody decreased the level of fractalkine in the culture media of HSC‐2 and YD10B OSCC cells. Treatment with a fractalkine‐neutralizing antibody reduced TGF‐β‐stimulated invasion by HSC‐2 and YD10B cells. Fractalkine treatment increased the viability, invasion, and uPA secretion of both OSCC cell lines. Furthermore, OSCC cell bone invasion was assessed following subcutaneous inoculation of wild‐type or TGF‐β knockdown OSCC cells in mouse calvaria. TGF‐β knockdown prevented erosive bone invasion, reduced the number of osteoclasts at the tumor‐bone interface, and downregulated fractalkine expression in mouse tumor tissues. Our results indicate that the production of fractalkine is stimulated by TGF‐β and mediates TGF‐β‐induced cell invasion in several OSCC cell lines showing an erosive pattern of bone invasion. Fractalkine may be a useful predictive marker and therapeutic target for OSCC‐induced bone destruction.     

The role of hypoxia in oral cancer and potentially malignant disorders: a review

Oral and oropharyngeal cancer are major health problems globally with over 500 000 new cases diagnosed annually. Despite the fact that oral cancer is a preventable disease and has the potential for early detection, the overall survival rate remains at around 50%. Most oral cancer cases are preceded by a group of clinical lesions designated ‘potentially malignant disorders’. It is difficult to predict if and when these lesions may transform to malignancy, and in turn it is difficult to agree on appropriate management strategies. Understanding underlying molecular pathways would help in predicting the malignant transformation of oral potentially malignant disorders and ultimately identifying effective methods for early detection and prevention of oral cancer. Reprogramming energy metabolism is an emerging hallmark of cancer that is predominantly controlled by hypoxia‐induced genes regulating angiogenesis, tumour vascularization, invasion, drug resistance and metastasis. This review aims to highlight the role of hypoxia in oral carcinogenesis and to suggest future research implications in this arena.     

Effects of interleukin‐1β and tumor necrosis factor‐α on macrophage inflammatory protein‐3α production in synovial fibroblast‐like cells from human temporomandibular joints

Background

Interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α) are key mediators of the intracapsular pathological conditions of the temporomandibular joint (TMJ). Therefore, the gene expression profiles in synovial fibroblast‐like cells (SFCs) from patients with internal derangement of the TMJ were examined after they were stimulated with IL‐1β or TNF‐α to determine which genes were altered.

Methods

Ribonucleic acid was isolated from SFCs after IL‐1β or TNF‐α treatment. Gene expression profiling was performed using oligonucleotide microarray analysis. On the basis of the results of this assay, we investigated the kinetics of macrophage inflammatory protein‐3α (MIP‐3α) gene expression using PCR, and protein production in TMJ SFCs stimulated by IL‐1β or TNF‐α using an ELISA. Inhibition experiments were performed with MAPK and NFκB inhibitors. SFCs were stimulated with IL‐1β or TNF‐α after treatment with inhibitors. The MIP‐3α levels were measured using an ELISA.

Results

Macrophage inflammatory protein‐3α was the gene most upregulated by IL‐1β‐ or TNF‐α stimulation. The mRNA and protein levels of MIP‐3α increased in response to IL‐1β in a time‐dependent manner. In contrast, during TNF‐α stimulation, the MIP‐3α mRNA levels peaked at 4 h, and the protein levels peaked at 8 h. In addition, the IL‐1β‐ and TNF‐α‐stimulated MIP‐3α production was potently reduced by the MAPK and NFκB signaling pathway inhibitors.

Conclusion

Interleukin‐1β and TNF‐α increased the MIP‐3α production in SFCs via the MAPK and NFκB pathways. These results suggest that the production of MIP‐3α from stimulation with IL‐1β or TNF‐α is one factor associated with the inflammatory progression of the internal derangement of the TMJ.     

Image processing analysis of oral cancer,oral potentially malignant disorders,and other oral diseases using optical instruments

Oral cancer screening is important for early detection and early treatment, which help improve survival rates. Biopsy is invasive and painful, while fluorescence visualization using optical instruments is non-invasive, convenient, and provides results in real time, and examinations can be repeated. The purpose of this study was to determine the usefulness of optical instruments in oral screening. A total of 314 patients who were examined using optical instruments at Tokyo Dental College between 2014 and 2018 were enrolled in this study. Fluorescence visualization images were analyzed using subjective and objective evaluations. Subjective evaluation for detecting oral cancer offered 98.0% sensitivity and 43.2% specificity. Regarding the objective evaluations for detecting oral cancer, sensitivity and specificity were 61.9% and 62.7% for mean luminance, 90.3% and 55.7% for luminance ratio, 56.5% and 67.7% for standard deviation of luminance, and 72.5% and 85.4% for coefficient of variation of luminance. Fluorescence visualization with subjective and objective evaluation using optical instruments is useful for oral cancer screening.