The dependence of breast cancer development on estrogen exposure explains the higher risk observed in women with history of early menarche, late menopause, and use of hormone replacement therapy. There are three suggested pathways explaining the carcinogenic effect of estrogen in the human breast, estrogen receptor mediated, direct genotoxic action and induction of aneuploidy. In the present work we are presenting evidence that epigenetic changes may be also involved in the estrogen induced neoplastic transformation. In order to determine whether the transformation of the human breast epithelial cells MCF-10F after treatment with 17-β-estradiol (E2) is mediated by epigenetic changes, we utilized Restriction Landmark Genomic Scanning (RLGS) for studying global CpG island methylation in an in vitro-in vivo model in which MCF-10F cells treated with E2 manifest loss of ductulogenesis (LoD) in collagen matrix as an early stage, progressing to increased invasiveness (INV) in an artificial reconstituted basement membrane, for culminating in tumorigenesis in a heterologous host (TUM) as the final stage of malignant transformation. Cells were collected at the three stages of progression described above for extraction of RNA and genomic DNA for determination of mRNA levels of expression by Real time RT-PCR and CpG island methylation by RLGS, respectively. Our results revealed that in 13 genes associated with branching morphogenesis the expression of mRNA and methylation status varied at each one of the stages of cell transformation. Cells in the LoD stage contained 3 hypermethylated genes that included HOXA9, SNIP and BMP6, and one hypomethylated gene, CAST. In INV cells HOX9, and additional six genes including SPRY1 were hypermethylated. In TUM cells HOXA9 and NRG1 and four additional genes, including EPB49 and FOXD1 that were also hypermethylated in INV cells, were detected. Our data indicate that the inactivation of genes controlling the processes of branching and morphogenesis plays an important role in the early stages of estrogen-induced transformation, and that silencing by CpG island methylation recruits additional genes contributing to the loss of normal pattern of mammary ductal tree formation, increased invasiveness and finally tumor formation. (This work was supported by RO1-CA094098 and DAMD17-03-1-0247).

[Proc Amer Assoc Cancer Res, Volume 47, 2006]