Promoter methylation of P16, RARbeta, E-cadherin, cyclin A1 and cytoglobin in oral cancer: quantitative evaluation using pyrosequencing

Abstract

Methylation profiling of cancer tissues has identified this mechanism as an important component of carcinogenesis. Epigenetic silencing of tumour suppressor genes through promoter methylation has been investigated by a variety of means, the most recent of which is pyrosequencing. We have investigated quantitative methylation status in oral squamous cell carcinoma patients. Fresh tumour tissue and normal control tissue from resection margin was obtained from 79 consecutive patients undergoing resection of oral squamous cell carcinoma. DNA was extracted and bisulphite treated. PCR primers were designed to amplify 75-200 bp regions of the CpG rich gene promoters of p16, RARbeta, E-cadherin, cytoglobin and cyclinA1. Methylation status of 4-5 CpG sites per gene was determined by pyrosequencing. Significant CpG methylation of gene promoters within tumour specimens was found in 28% for p16, 73% for RARbeta, 42% for E-cadherin, 65% for cytoglobin and 53% for cyclinA1. Promoter methylation was significantly elevated in tumours compared to normal tissue for p16 (P = 0.048), cytoglobin (P = 0.002) and cyclin A1 (P = 0.001) but not in RARbeta (P = 0.088) or E-cadherin (P = 0.347). Concordant methylation was demonstrated in this tumour series (P = 0.03). Significant differences in degree of methylation of individual CpG sites were noted for all genes except RARbeta and these differences were in a characteristic pattern that was reproduced between tumour samples. Cyclin A1 promoter methylation showed an inverse trend with histological grade. Promoter methylation analysis using pyrosequencing reveals valuable quantitative data from several CpG sites. In contrast to qualitative data generated from methylation specific PCR, our data demonstrated p16 promoter methylation in a highly tumour specific pattern. Significant tumour specific methylation of cyclin A1 promoter was also seen. Cytoglobin is a novel candidate tumour suppressor gene highly methylated in upper aero-digestive tract squamous cancer.

Figure 1

PCR and sequencing primers for the cytoglobin gene promoter.

Figure 2

Representative pyrograms for RARβ. The four targeted cytosines are enclosed in unshaded squares (as reverse strand was read, G peaks (arrowed) indicate methylated cytosine while (A) indicates unmethylated cytosine). The control, non-CpG cytosine residue showing complete conversion of cytosine to uracil by bisulphite treatment is shown in the left-hand shaded box. Normal tissue (top panel) demonstrates no methylation while tumour tissue (bottom panel) demonstrates a significant level of methylation at all four target bases. The Methylation Index (MtI) is calculated as the average rate of G incorporation at each CpG.

Figure 3

Box and whisker plots of methylation Indices for the five genes studied. Boxes include 50% data, O=Outlier ‘cases with values between 1.5 and 3 box lengths from the upper or lower edge of the box. The box length is the interquartile range.’ ^*=Extreme ‘cases with values more than three box lengths from the upper or lower edge of the box’.

Figure 4

CpG methylation patterns within the promoter regions of the five genes studied.