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.

Cervical cancer risk in HPV‐positive women after a negative FAM19A4/mir124‐2 methylation test: A post hoc analysis in the POBASCAM trial with 14 year follow‐up

“…DNA from cervical scrapes was isolated using the Nucleo‐Spin 96 Tissue kit (Macherey‐Nagel, Duren, Germany) and a Microlab Star robotic system (Hamilton, Planegg, Germany) according to manufacturers’ protocol 32. Extracted DNA was subjected to bisulphite treatment using the EZ DNA Methylation Kit (Zymo Research, Irvine, CA) as described previously 33, 34. Bisulphite‐converted DNA was used as template for DNA methylation analysis for FAM19A4 and mir124‐2 genes using a prototype version of the QIAsure Methylation Test (Qiagen, Hilden, Germany) and Rotorgene PCR‐system (Qiagen).…”

Clinical implications of (epi)genetic changes in HPV-induced cervical precancerous lesions

“…HPV 68 has been classified as probable high-risk (also known as IARC class 2A) and another seven types have been classified as possible high-risk (HPV 26,53,66,67,70,73 and 82;also known as IARC class 2B) 5 .…”

Aberrant methylation of the CADM1 promoter is associated with poor prognosis in hepatocellular carcinoma treated with liver transplantation

“…the expressions of cADM1 protein in 56 paired Hcc tissues were detected by immunohistochemistry assay as described previously (20). slides were incubated overnight at 4˚C with rabbit anti-human polyclonal antibody against the c-terminus of cADM1 (1:400 dilution; tBs/5% FBs/0.1% triton X-100; Abcam), and then stained with 3,3'-diaminobenzdine.…”