Association between dense CADM1 promoter methylation and reduced protein expression in high-grade CIN and cervical SCC

Abstract

We previously showed that silencing of TSLC1, recently renamed CADM1, is functionally involved in high-risk HPV-mediated cervical carcinogenesis. CADM1 silencing often results from promoter methylation. Here, we determined the extent of CADM1 promoter methylation in cervical (pre)malignant lesions and its relation to anchorage-independent growth and gene silencing to select a CADM1-based methylation marker for identification of women at risk of cervical cancer. Methylation-specific PCRs targeting three regions within the CADM1 promoter were performed on high-risk HPV-containing cell lines, PBMCs, normal cervical smears, and (pre)malignant lesions. CADM1 protein expression in cervical tissues was analysed by immunohistochemistry. All statistical tests were two-sided. Density of methylation was associated with the degree of anchorage-independent growth and CADM1 gene silencing in vitro. In cervical squamous lesions, methylation frequency and density increased with severity of disease. Dense methylation (defined as >or= 2 methylated regions) increased from 5% in normal cervical samples to 30% in CIN3 lesions and 83% in squamous cell carcinomas (SCCs) and was significantly associated with decreased CADM1 protein expression (p < 0.00005). The frequency of dense methylation was significantly higher in >or= CIN3 compared with <or= CIN1 (p = 0.005), as well as in SCCs compared with adenocarcinomas (83% versus 23%; p = 0.002). Detection of dense CADM1 promoter methylation will contribute to the assembly of a valuable marker panel for the triage of high-risk HPV-positive women at risk of >or= CIN3.