Correlation Of Deregulated Like-Acetylglucosaminyltransferase And Aberrant α-dystroglycan Expression With Human Tongue Cancer Metastasis

Oral squamous cell carcinoma is the sixth most common malignancy worldwide. In China,21000cases of oral cancer are diagnosed each year, and approximately11000patients die of the disease and often invades surrounding tissues and metastasizes to cervical lymph nodes. Tongue cancer is the most common malignancy diagnosed within the oral cavity. To reduce the rate of lymph node metastasis and improve the survival rates of oral squamous cell carcinomas patients becomes very urgent and necessary.Abnormal cellular interaction with the extracellular matrix (ECM) is a hallmark of malignant transformation. Dystroglycan (DG) is a non-integrin adhesion molecule, DG was expressed predominantly on the surfaces between cells and basement membrane, acting as an important "molecular bridge" of cell-ECM interaction. DG has been reported to play an important role in regulating basal membrane morphogenesis, cytoskeletal organization, cell polarization, cell growth, signal transduction, and maintenance of tissue integrity in epithelial cells, which might implicate DG in cancer biology. Aberrant expression of a-DG has been found in many epithelium-derived cancers, suggesting that this molecule might play an important role in cancer development and progression. However, relatively little is known about the underlying mechanisms of aberrant a-DG expression in cancers. Hitherto, seven glycosyltransferases or glycosyltransferase-like genes have been found to be involved in a-DG functional glycosylation. LARGE plays a key role in post-translational modulating both a-DG glycosylation and functional expression. Therefore, the purpose of the present study was to investigate whether the LARGE gene inactivation which then lead to abrrent expression and glycosylation of a-DG, and research the underlying mechanisms in tongue cancer.Part One Glycosylated a-DG expression was reduced or absent in human tongue cancer and correlated with nodal metastasisObjective:To examine the expression of a-DG and the relationships with clincopathological features in tongue cancer. Materials and Methods: Immunohistochemistry was employed to analyze the protein expression levels of a-DG in fifty tongue cancer tissues and thirteen normal oral mucosa samples. The results were compared to the cliniopathological parameters. Western blot was used to detect the expression levels of a-DG in tongue carcinoma cell lines (CAL27and SCC4), and the mRNA of DG was exramined by RT-PCR. Results:In normal epithelium, all types of a-DG (6C1, VIA4-1and IIH6) were found to localize in the basal cell layers of the squamous epithelium. thea-DG (6C1) was detected in tongue cancer, while a-DG (VIA4-1) and a-DG (IIH6) were nearly absent in all carcinoma samples. The loss of VIA4-1or IIH6expression was statistically correlated with TNM stages (N=50, P=0.005and0.013respectively) and lymph node metastasis (P=0.016and0.025respectively). a-DG (VIA4-1) and a-DG (IIH6) were undetected in cancer cell lines, DG mRNA was clearly detectable in both cell lines. Conclusion: The glycan-epitopes of a-DG were reduced even lost in tongue cancer, and a-DG was not functionally glycosylated in both CAL27and SCC4. It suggested that loss of a-DG protein in tongue cancer may be due to a post-translational modification rather than an expression or trafficking defect.Part Two Deregulation of LARGE expression and hypermethylation of the LARGE gene promoter were correlated with nodal metastasis and aberrant a-DG glycosylation in tongue cancerObjective:To detect the expression of LARGE protein and the methylation stutas of the LARGE promoter in tongue cancer. Analysis the relationship between loss of a-DG glycosylation and the hypermethylation of LARGE promoter. Materials and Methods:In fifty tongue cancer tissues and thirteen normal oral mucosa samples, immunohistochemistry was employed to analyze the protein expression levels of LARGE, and MSP was used to detect the methylation stutas of the LARGE promoter, the results were compared to the cliniopathological parameters. Western blot was used to detect the expression levels of LARGE cancer cell lines, and the LARGE mRNA was exramined by RT-PCR. Results:LARGE protein was obviously expressed in normal controls, but decreased or lost in tongue cancer. Hypermethylation of the LARGE gene promoter was more frequent in human tongue cancer (32/50) than that in normal mucosa tissues (3/13)(P=0.01). Both the reduced expression of LARGE and hypermethylation of the LARGE gene promoter was significantly related to the clinical staging (P=0.022and0.015respectively) and lymph node status (P=0.000and0.043respectively). Furthermore, both CAL27and SCC4cells were hypermethylated on LARGE gene promoter. There was statistically correlation between loss of α-DG glycosylation and hypermethylation of the LARGE gene promoter in tongue cancer. Conclusion:Epigenetic silence of LARGE is a potential mechanism underlying the failure of a-DG post-translational modification in the formation and metastasis of tongue cancer.Part Three LARGE overexpression and Aza-dC treatment restored a-DG glycosylationObjective:To determine whether a-DG glycosylation can be restored by overexpressing recombinant LARGE and treated with demethylating agent5-aza-2’-deoxycytidine (Aza-dC) in cancer cell lines. Materials and Methods: Constructed recombinant LARGE expression plasmids, then transfected the plasmids into the cancer cells, or treated the cancer cells with demethylating agent Aza-dC. Subsequently, LARGE mRNA and protein were detected by RT-PCR and western blot, then detected for DG mRNA and a-DG protein. Results:LARGE mRNA and protein expression were increased in the cells which transfection and treated with Aza-dC. What is more, the glycan-epitopes of a-DG were successfully rescued by LARGE transfection and Aza-dC treatment. on the contrary, there was no obvious difference of DG mRNA or expression of the a-DG core protein between control and LARGE-transfected CAL27and SCC4cells. Conclusion:Treatment with demethylating agents or LARGE overexpression could restore LARGE rxpression, and then lead to rescue functional glycosylation of a-DG in tongue cancer cell lines.Part Four LARGE overexpression and Aza-dC treatment resulted in laminin binding enhancement and migration suppression of tongue cancer cellsObjective:To determine whether the functional a-DG glycosylation can be restored by overexpressing recombinant LARGE in cancer cell lines. Materials and Methods: After transfected the plasmids or treated with Aza-dC, laminin overlay assays were performed to exramine the ability between laminin binding and a-DG, and a cell adhesion assays were performed to detecte the ability of cells binding to basement membrane protein, the migration capacity of cells was performed by Transwell assay. Results:LARGE-overexpressed and Aza-dC-treated tongue cancer cells increaseda-DG binding with laminin ligand compared to control group, and cells adhere to laminin more effectively. In addition, Transwell assay showed that there was a significant decrease in migration of CAL27and SCC4cells with Aza-dC treatment (or cells with forced expression of LARGE) compared with control. Conclusion:The results indicates that LARGE deregulation-induced loss of a-DG impairs the ability of the cell to escape from its laminin anchor and thereby increases its ability of migration. And LARGE-overexpressed and Aza-dC-treated could reverse the process.In summary, the present study found that both loss of a-DG expression and LARGE deregulation were correlated with and TNM stage and nodal metastasis of human tongue cancer. We also confirmed that LARGE hypermethylation was associated with the aberrant a-DG glycosylation, and that LARGE overexpression or demethylation could restore a-DG glycosylation. The results revealed a close correlation of deregulated LARGE and aberrant a-DG expression with human tongue cancer metastasis, and loss of functional glycosylation of α-DG is attributed to epigenetic silencing of LARGE.