Deregulation of inhibitors of DNA binding in human breast cancer cells with acquired resistance to 4-hydroxytamoxifen and fulvestrant

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Members of the inhibitor of DNA binding or differentiation (ID) protein family play a central role in the control of cell differentiation and proliferation. Generally, constitutive expression of ID proteins blocks cell differentiation and drives proliferation. The primary mechanism of action of this protein family is inhibition of basic helix-loop-helix transcription factor activity, and ID proteins have recently been implicated in the regulation of normal and transformed mammary epithelial cell phenotypes. However, the role of IDs in regulating breast cancer cell proliferation remains unclear. In this study, we investigated whether ID expression is regulated by 17β-estradiol (E2) in human breast cancer cells, and whether the expression of IDs is altered in cells with acquired resistance to the antiestrogens 4-hydroxytamoxifen (OHT, the active metabolite of tamoxifen) and fulvestrant (ICI 182,780, an estrogen receptor downregulator). Antiestrogen-resistant cells, MCF7-OHT^R and MCF7-ICI^R, were established by growing hormone-sensitive MCF7 cells in hormone-free medium supplemented with OHT or ICI 182,780. Estrogen receptor alpha (ER) protein levels were similar in MCF7-OHT^R and MCF7 cells, and MCF7-OHT^R cells remained responsive to fulvestrant, but to a less extent compared to MCF7 cells. A low level of ER was observed in MCF7-ICI^R cells (∼2% relative to MCF7), and these cells were cross-resistant to OHT. MCF7, MCF7-OHT^R and MCF7-ICI^R cells were treated with vehicle (DMSO) or E2 (10 nM, 4 h), and global changes in gene expression were determined using Affymetrix Human Genome U133 Plus 2.0 Array, performed in quadruplicate. Microarray results were validated by real-time quantitative PCR. Changes in the mRNA levels of four known ID family members (ID1, ID2, ID3 and ID4) were observed. The results showed that E2 treatment decreased (>2-fold; p<0.01) the mRNA levels of all four IDs in both parental MCF7 and MCF7-OHT^R but not in MCF7-ICI^R cells. Compared to MCF7 cells, the basal expression levels of IDs were differentially altered in MCF7-OHT^R and MCF7-ICI^R cells. Basal ID1 mRNA level was increased (>2-fold; p<0.01) in both MCF7-OHT^R and MCF7-ICI^R cells, while basal ID3 and ID4 mRNA levels were increased (>2-fold, p<0.01) only in MCF7-OHT^R cells. The level of ID2 mRNA was decreased (>2-fold, p<0.01) in MCF7-ICI^R cells only. These results demonstrate that IDs are E2-responsive genes in MCF7 cells. Furthermore, ID expression patterns in breast cancer cells become differentially altered during acquired resistance to OHT or fulvestrant. We are currently investigating the effects of gain- or loss-of-function of IDs on the response of breast cancer cells to E2 and antiestrogens.