The Wilms' tumor suppressor WT1 induces estrogen-independent growth and anti-estrogen insensitivity in ER-positive breast cancer MCF7 cells

Abstract

A switch from estrogen-dependent to estrogen-independent growth is a critical step in malignant progression of breast cancer and is a major problem in endocrine therapy. However, the molecular mechanisms underlying this switch remain poorly understood. The Wilms' tumor suppressor gene, wt1, encodes a zinc finger protein WT1 that functions as a transcription regulator. High levels of the WT1 expression have been associated with malignancy of breast cancer. The goal of this study was to investigate the function of WT1 in malignant progression of breast cancer. We found that the high passage ER-positive breast cancer MCF7H cells expressed EGFR, HER2 and WT1 at higher levels compared to the low passage MCF7L cells. MCF7H cells responded weakly to estrogen stimulation, grew rapidly in the absence of estrogen and were insensitive to anti-estrogens such as ICI 182,780 and 4-hydroxy-tamoxifen (4OH-TAM). We also established stable cell lines from the low passage MCF7L cells to constitutively express exogenous WT1 and found elevated levels of EGFR and HER2 expression, estrogen-independent growth and anti-estrogen insensitivity in WT1-transfected MCF7L cells. These results suggested WT1 promotes estrogen-independent growth and anti-estrogen resistance in ER-positive breast cancer cells presumably through activation of the signaling pathways mediated by the members of EGFR family.

Figure 1

High passage MCF7 cells (MCF7^H) exhibit estrogen-independent and anti-estrogen insensitive growth. (A). Growth rate of MCF7^H and MCF7^L cells. Cells maintained in steroid-free medium were seeded at 1×10⁴ cells per well in 6-well plates, incubated in medium containing DMSO (V) or 1 nM of 17β-estradiol (E2) and counted every other day. Data presented are means of three independent experiments; bars, SE. (B) Cells were seeded at 1×10⁴ cells per well in 60-mm dishes, incubated in medium containing DMSO (V), 1 nM of 17β-estradiol (E2), 1 μM 4OH-TAM (4OH) or 1 μM ICI 182,780 (ICI) and counted in 12 days. Column, means of three independent experiments; bars, SE. a and b, P<0.01 vs. cells in the absence E2; c and d, P<0.01 vs. MCF7L cells treated with anti-estrogens.

Figure 2

High passage MCF7^H cells express increased levels of EGFR, HER2 and WT1. Western blot analysis of the cell lysates from MCF7^H cells and MCF7^L cells.

Figure 3

Lapatinib and 4-hydroxytamoxifen inhibit ER-mediated transcription activity in MCF7^H cells. MCF7^H cells and MCF7^L cells were transfected with p2X ERE-Luc reporter plasmid for 36 h before treatment with DMSO (V), 1 μM 4OH-TAM (4OH), 5 μM Lapatinib or both drugs together in the absence or presence of 1 nM of E2 for an additional 12 h. Data are shown as means ± SE from three independent experiments. The value of ERE luciferase activity in vehicle-treated MCF7^L cells is set as 1.

Figure 4

Cyclin D1 and c-Myc expression is regulated by the MAPK/ERK pathway in MCF7^H cells. (A). Western blot analysis of the lysates from MCF7^H cells and MCF7^L cells treated with 1 nM of E2 for different period of time as indicated using anti-cyclin D1 and c-Myc antibodies. (B). Western blot analysis of the lysates from MCF7^H cells and MCF7^L cells cultured in normal medium containing 5% FCS or in medium containing 5% dextran-charcoal-stripped FCS containing DMSO vehicle (V), 1 μM 4OH-TAM (4OH), 1 μM ICI 182,780 (ICI) or 10 μM UO126 for 12 h.

Figure 5

Constitutive expression of recombinant WT1 in MCF7^L cells induces protein levels of EGFR and HER2. (A and B) Western blot analysis of the lysates from parental MCF7^L cells (MCF7/P), MCF7^L cells transfected with the empty expression vector (MCF7/V) and clonal cell lines from MCF7^L cells transfected with WT1 expression vector (MCF7/WT1-2, -3 and -4).

Figure 6

WT1-transfected MCF7^L cells exhibit estrogen-independent and anti-estrogen insensitive growth (A). Growth rate of MCF7^L cells transfected with the empty expression vector (MCF7/V) and clonal cell lines from MCF7^L transfected with WT1 expression vector (MCF7/WT1-2, -3 and -4). Cells maintained in normal medium containing 5% FCS were seeded at 1×10⁴ cells per well in 6-well plates and counted every other day. Data are shown as means ± SE from three separate experiments (B). Cells were seeded at 1×10⁴ cells per 60-mm dishes, incubated in medium containing DMSO (V), 1 nM of 17β-estradiol (E2), 1 μM 4OH-TAM (4OH) or 1 μM ICI 182,780 (ICI) and counted at day 12. Column, means of three independent experiments; bars, SE. a–c, P<0.01 vs. MCF7/V cells; d–f, P<0.01 vs. MCF7/V cells.