ABSTRACT: Estrogen is a risk factor for breast cancer, yet its mechanism in the initiation of this disease is not clear. Here we use a model of estrogen-mediated malignant transformation of MCF-10F cells to identify the temporal acquisition of changes in genome structure and gene expression that correspond to the progressive transformed phenotype culminating in tumorigenesis. Genomic DNA and total RNA were isolated from MCF10F, trMCF (MCF10F transformed by 70 nM 17-beta estradiol), bsMCF (trMCF selected by Boyden chamber, tumorigenic), bcMCF(clones of bsMCF), and caMCF (cells from tumors of bsMCF grown in SCID mice). The Affymetrix 100k SNP and HG-U133_Plus_2 chips were used for genotyping and gene expression analyses. Changes in chromosomal copy number and loss of heterozygosity were progressive. Gross changes were rarely observed in the trMCF10F cells; the earliest was a gain in chromosome 1p, 1p36.12-1p36.21. In the bsMCF and their sub-clones bcMCF, additional gains were seen in chromosomes 1p, 1p36.12-1pter, 5q, 5q21.1-5q35.3, and 13q, 13q21.32-13q34; losses were detected in chromosomes 4 and 8p, 8p11.1-8p23.1. In the caMCF, additional losses were seen in chromosomes 3p, 3p12.1-3p14.1, 9p, 9p22.1-9p24.3, and 18q, 18q11.2-18q23. Using microarray analysis we were able, in some cases, to narrow the regions of interest and their associated candidate cancer genes. The observation of progressive genomic changes, along with the tumor phenotype of a poorly differentiated adenocarcinoma, indicate an underlying mechanism common to breast cancer development in humans, and adds to the evidence indicating that estrogens are mutagenic and contribute early to the process of breast cancer. Experiment Overall Design: Human MCF-10F cells were transformed by 70nM 17-beta-estradiol. The transformed cells were selected by Boyden chamber. The selected cells were injected into SCID mice and also cloned and expanded. The thus grown tumors were harvested for cell culture. Genomic DNA and total RNA were isolated from cell lines in each step for 100K SNP and gene expression microarray assay.
