Abstract
INTRODUCTION: Breast cancer metastasis is a complex, multi-step biological
process. Genetic mutations along with epigenetic alterations in the
form of DNA methylation patterns and histone modifications contribute
to metastasis-related gene expression changes and genomic instability.
So far, these epigenetic contributions to breast cancer metastasis
have not been well characterized, and there is only a limited understanding
of the functional mechanisms affected by such epigenetic alterations.
Furthermore, no genome-wide assessments have been undertaken to identify
altered DNA methylation patterns in the context of metastasis and
their effects on specific functional pathways or gene networks. METHODS:
We have used a human gene promoter tiling microarray platform to
analyze a cell line model of metastasis to lymph nodes composed of
a poorly metastatic MDA-MB-468GFP human breast adenocarcinoma cell
line and its highly metastatic variant (468LN). Gene networks and
pathways associated with metastasis were identified, and target genes
associated with epithelial-mesenchymal transition were validated
with respect to DNA methylation effects on gene expression. RESULTS:
We integrated data from the tiling microarrays with targets identified
by Ingenuity Pathways Analysis software and observed epigenetic variations
in genes implicated in epithelial-mesenchymal transition and with
tumor cell migration. We identified widespread genomic hypermethylation
and hypomethylation events in these cells and we confirmed functional
associations between methylation status and expression of the CDH1,
CST6, EGFR, SNAI2 and ZEB2 genes by quantitative real-time PCR. Our
data also suggest that the complex genomic reorganization present
in cancer cells may be superimposed over promoter-specific methylation
events that are responsible for gene-specific expression changes.
CONCLUSION: This is the first whole-genome approach to identify genome-wide
and gene-specific epigenetic alterations, and the functional consequences
of these changes, in the context of breast cancer metastasis to lymph
nodes. This approach allows the development of epigenetic signatures
of metastasis to be used concurrently with genomic signatures to
improve mapping of the evolving molecular landscape of metastasis
and to permit translational approaches to target epigenetically regulated
molecular pathways related to metastatic progression.
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