Significant changes in gene expression and DNA methylation status likely occur as breast cancer cells acquire resistance to antiestrogens. We therefore undertook a comparative gene expression and DNA methylation analysis of the hormone-sensitive breast cancer MCF7 cell line and MCF7 cell derivatives that acquired resistance to 4-hydroxytamoxifen (OHT) and fulvestrant (ICI 182,780). Antiestrogen-resistant cells, OHTR and ICIR, were established by long-term culture of MCF7 cells in hormone-free medium supplemented with OHT or ICI 182,780. Estrogen receptor alpha (ER) protein levels were comparable in OHTR and MCF7 cells, and OHTR remained responsive to growth inhibition by ICI 182,780, but to a lesser extent compared to MCF7. In contrast, ICIR cells had low ER levels (∼2% relative to MCF7) and were cross-resistant to OHT. Global gene expression profiles were determined using Affymetrix Human Genome U133 Plus 2.0 Arrays; expression of individual genes of interest was validated by real-time quantitative PCR. Genomic DNA methylation status was analyzed by differential methylation hybridization of microarrays containing 12,288 CpG island (CGI) fragments. Pairwise comparisons of gene expression patterns in cells treated with vehicle (DMSO) or 17β-estradiol (E2, 10 nM, 4 h) revealed that regulation of E2-responsive genes was partially preserved in OHTR but largely lost in ICIR cells. In MCF7 cells, a total of 285 genes were E2-responsive (up- or down-regulated, >2-fold; P<0.01); among these, 68 genes (25%) and 1 gene (0.4%) remained responsive to E2 in OHTR and ICIR, respectively. In addition, in OHTR cells, 61 genes gained E2-responsiveness. Global gene expression profiling analysis further revealed that basal expression levels of 320 and 2,730 genes were altered (>3 fold vs MCF7, P<0.01) in OHTR and ICIR cells, respectively. Among the altered genes, only 111 genes (2%) were commonly up- or down-regulated in both OHTR and ICIR cell lines. Based on a biological function/pathways analysis, we observed differential deregulation of cell regulatory pathways in OHTR and ICIR cells. For instance, interferon-induced genes and ephrin receptors were prominently up-regulated in ICIR but not in OHTR cells. Based on genomic DNA methylation analysis, ICIR cells showed a significant increase in number of methylated CGIs (361 in ICIR vs. 116 in MCF7), whereas DNA methylation patterns were similar in OHTR and MCF7 cells. Taken together, this study revealed that gene expression pattern and DNA methylation status were differentially altered by OHT and fulvestrant in breast cancer cells. Genes that were commonly or distinctly changed in ICIR and OHTR cells will be further evaluated as potential targets for therapeutic approaches of endocrine-resistant tumors.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]