Methylation at HPV18 E2 binding sites increases during cervical carcinogenesis

Abstract

BACKGROUND: There are four E2 binding sites (E2BSs) at the LCR region of HPV18 genome. Binding of E2 protein to different E2BSs may exert either transcriptional activation/repression on E6 and E7 oncoprotein, depending on their distance from the TATA box of the promoter. Methylation status at the E2BSs may affect the relative binding of E2 protein to them and may change during cervical carcinogenesis. OBJECTIVES. To investigate and compare the methylation status of E2BSs 1, 2 and 4 in HPV18-positive cervical cancer, high-grade CIN, low-grade CIN/ASCUS and normal cervical epithelium. METHODS. DNA was extracted from paraffin sections of 24 cases of HPV18-positive cervical cancer, as well as from cytology swabs / paraffin sections of 11 cases of HPV18-positive high-grade CIN, 12 cases of low-grade CIN/ ASCUS and 7 cases of normal cervical epithelium. Methylation percentage of individual CpGs at E2BS 1, 2 and 4 were assessed by pyrosequencing. The average percentage of CpG methylation at each E2BS was calculated and compared among different histological groups. RESULTS. HPV18 E2BS 1&2 remains more methylated than E2BS 4 in all histological groups. Both HPV18 E2BS 1&2 and E2BS 4 methylation increases significantly from ASCUS/ low-grade CIN to high-grade CIN (2.7% to 6.9% , p=0.019 and 1.2% to 6.7%, p=0.001 respectively) and then further increases form high-grade CIN to cervical cancer (6.9% to 55.4%, p<0.001 and 6.7% to 23.4%, p=0.005 respectively). CONCLUSIONS. Differential methylation at different E2BSs was observed in HPV18-postive cervical cancer, cervical premalignant lesions and normal cervical epithelium. Increasing methylation at HPV18 E2BSs is a potentially useful adjunctive molecular marker for predicting progression from ASCUS / low-grade CIN to high-grade CIN and from high-grade CIN to cervical cancer.