Dissecting complex epigenetic alterations in breast cancer using CpG island microarrays

It is now clear that aberrant DNA methylation observed in cancer cells is n ot restricted to a few CpG islands, but affects multiple loci. When this ep igenetic event occurs at the 5'-end of the regulatory region of genes, it i s frequently associated with transcriptional silencing. To investigate furt her this widespread event in the tumor genome, we developed a novel microar ray containing 7776 short GC-rich tags tethered to glass slide surfaces. Th is DNA chip was used to study 17 paired tissues of breast tumors and normal controls. Amplicons, representing differential pools of methylated DNA fra gments between tumors and normal controls, were cohybridized to the microar ray panel. Hypermethylation of multiple CpG island loci was then detected i n a two-color fluorescence system. Approximately 1% (on average, 83 loci) o f these CpG islands examined were hypermethylated in this patient group. Hi erarchical clustering segregated these tumors based on their methylation pr ofiles and identified a group of CpG island loci that corresponds to the ho rmone-receptor status of breast cancer. This observation was independently confirmed by examining a single locus, the promoter of the human glypican 3 gene, which was predominately hypermethylated in the hormone receptor-nega tive tumors. Our findings support the notion that hypermethylation of criti cal CpG island loci influences cancer development and produces distinct epi genetic signatures for particular tumor subtypes.