DNA methylation is a fundamental epigenetic event associated with physiologic and pathologic conditions including cancer. Hypermethylation of CpG islands at active gene promoters lead to transcriptional repression while hypomethylation is associated with gene overexpression. The aim of this study was to identify genes in adenoid cystic carcinoma (ACC) of salivary gland strongly deregulated by epigenetic CpG island methylation, to validate selected genes by conventional techniques, and to correlate the findings with clinico-pathologic factors.
We analyzed 16 matched normal and tumor tissues for aberrant DNA methylation using the methylated CpG island amplification and microarray (MCAM) method, and the pyrosequencing technique.
Microarray analysis showed hypomethylation in seven, and hypermethylation in 32 CpG islands. Hypomethylation was identified in CpG islands near FBXO17, PHKG1, LOXL1, DOCK1 and PARVG. Hypermethylation was identified near genes encoding predominantly transcription factors (EN1, FOXE1, GBX2, FOXL2, TBX4, MEIS1, LBX2, NR2F2, POU3F3, IRX3, TFAP2C, NKX2-4, PITX1, NKX2-5), and 13 genes with different functions (MT1H, EPHX3, AQPEP, BCL2L11, SLC35D3, S1PR5, PNLIPRP1, CLIC6, RASAL, XRN2, GSTM5, FNDC1, INSRR). Four CpG islands by EN1, FOXE1, TBX4, and PITX1 were validated by pyrosequencing.
The highly methylated genes in tumor versus normal are linked to developmental, apoptotic and other fundamental cellular pathways, suggesting that downregulation of these genes is associated with ACC development and progression. With EN1 hypermethylation showing potential as possible biomarker for ACC in salivary gland, the biological and therapeutic implications of our findings require further preclinical investigations.