Role of Promoter Methylation in Regulating E-cadherin Gene Expression in Oral Squamous Cell Carcinoma Cells

Cadherins are cell-cell adhesion molecules that modulate the epithelial phenotype and can regulate tumor invasion in oral squamous cell carcinoma (SCC). Objectives: To identify the possible mechanism responsible for the loss of cadherin expression in oral SCC. Methods: We used Western blotting to screen for the expression of E- and N- cadherin in 14 oral SCC cell lines. Next, RT-PCR was used to amplify the total RNA and PCR to amplify the genomic DNA. Then, methylation-specific PCR (MSP) was used to screen for the promoter of the E-cadherin gene in these cell lines. Finally, the methylation- positive lines were treated with a DNA methylation inhibitor (5-aza-2-deoxycitidine, a cytidine analog), and subsequently analyzed by Western blotting, immunofluorescence, and immunoprecipitation. Results: Two (HA376, HOC313) of 14 cell lines were negative for the expression of E-cadherin. However, the expression of N-cadherin in these two cell lines was strongly expressed while the remainder of cell lines were negative for this receptor. For the two E-cadherin-negative cell lines, PCR amplified the E-cadherin gene fragments from the genomic DNA, but RT-PCR failed to amplify fragments of E-cadherin from the total RNA. MSP analysis clearly indicated that the E-cadherin promoters in these two cell lines were extensively methylated. After the demethylation treatment, both Western blotting and immunofluorescence staining showed that HA376 cells had re-expressed E-cadherin, and this was confirmed by immunoprecipitation. Conclusions: The results suggest that 5` CpG island methylation of E-cadherin promoter regulates the expression of this adhesion receptor in oral SCC. Additionally, N-cadherin expression appears to be reciprocally upregulated in oral SCC after the loss of E-cadherin expression by an as yet unknown mechanism. These events may play an important role in the regulation of tumor cell mobility and invasion. (This work was supported by NIH Grants P01 DE013904 and R01 DE011436).