Epigenetic alterations mapped with human promoter tiling microarrays identify genes implicated in epithelial-mesenchymal transition in a breast cancer metastasis model.

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Metastasis is a complex, multi-step biological process characterized by distinct yet interrelated steps that vary in their timing and efficiency. Genetic mutations alone may not be sufficient for metastasis progression, since altered epigenetic patterns of DNA methylation and histone modifications may also contribute to gene expression changes and genomic instability. To date, epigenetic contributions related to lymphatic metastasis are not well characterized. No genome-wide assessments have been performed to identify DNA methylation epimutations that could contribute to metastasis by inactivating tumour- or metastasis-suppressor genes, or lead to genomic instability. We undertook high-resolution DNA methylation profiling using Affymetrix human gene promoter tiling microarrays to identify metastasis-related DNA methylation changes associated with breast cancer metastasis to lymph nodes. We identified widespread hypo- and hypermethylation events in a highly metastatic variant (468LN cells) of a poorly metastatic MDA-MB-468 human breast adenocarcinoma cell line. Annotated gene lists were generated of the 2209 significantly hypermethylated and 1235 hypomethylated gene targets (p<0.05) identified in the 468LN cells. These data from the tiling array platform were integrated with the Partek Genomics Suite and Ingenuity Pathway Analysis software to investigate the biological relevance of the observed methylation changes. As well, we generated networks that addressed the biological relationships between genes implicated in epithelial-mesenchymal transition (EMT), tumour cell extravasation and migration. We overlaid our methylation data set onto these networks and confirmed functional associations between gene expression (by quantitative RealTime RT-PCR) and the DNA methylation status of a set of genes that included CDH1, Snai2, HEY1 and Twist. Our integrated approach provides a powerful whole-genome method to map epigenetic changes in tumourigenesis and can identify epigenetic targets that correlate with specific events in metastatic progression during breast cancer. We also describe the importance of validating specific epigenetic changes by using alternative methods of methylation analysis, as well as the necessity to functionally link specific epigenetic changes with gene expression. In this manner, identifying gene expression changes due to DNA methylation will permit translational approaches to target epigenetically-altered molecular pathways related to metatstatic progression. (This research was funded by the Canadian Breast Cancer Research Alliance Special Competition in New Approaches to Metastatic Disease.)