MicroRNA-221/222 confers breast cancer fulvestrant resistance by regulating multiple signaling pathways

Abstract

Fulvestrant is a selective estrogen receptor downregulator (SERD) and highly effective antagonist to hormone-sensitive breast cancers following failure of previous tamoxifen or aromatase inhibitor therapies. However, after prolonged fulvestrant therapy, acquired resistance eventually occurs in the majority of breast cancer patients, due to poorly understood mechanisms. To examine a possible role(s) of aberrantly expressed microRNAs (miRNAs) in acquired fulvestrant resistance, we compared antiestrogen-resistant and -sensitive breast cancer cells, revealing the overexpression of miR-221/222 in the SERD-resistant cell lines. Fulvestrant treatment of estradiol (E2)- and fulvestrant-sensitive MCF7 cells resulted in increased expression of endogenous miR-221/222. Ectopic upregulation of miR-221/222 in estrogen receptor-α (ERα)-positive cell lines counteracted the effects of E2 depletion or fulvestrant-induced cell death, thus also conferring hormone-independent growth and fulvestrant resistance. In cells with acquired resistance to fulvestrant, miR-221/222 expression was essential for cell growth and cell cycle progression. To identify possible miR-221/222 targets, miR-221- or miR-222- induced alterations in global gene expression profiles and target gene expression at distinct time points were determined, revealing that miR-221/222 overexpression resulted in deregulation of multiple oncogenic signaling pathways previously associated with drug resistance. Activation of β-catenin by miR-221/222 contributed to estrogen-independent growth and fulvestrant resistance, whereas TGF-β-mediated growth inhibition was repressed by the two miRNAs. This first in-depth investigation into the role of miR-221/222 in acquired fulvestrant resistance, a clinically important problem, demonstrates that these two 'oncomirs' may represent promising therapeutic targets for treating hormone-independent, SERD-resistant breast cancer.

Figure 1

Upregulation of miR-221/222 in breast cancer cell lines. (a) miR-221/222 expression in four antiestrogen-resistant MCF7 cell lines as measured by qRT–PCR analysis and relative to their expression level in parental MCF7 cells (mean±s.e., n = 3). **P<0.01. The following t-test results were obtained: MCF7-F, MCF7-Mek5 or MCF7-TNR versus MCF7-T, P<0.002. (b) Effect of E2, tamoxifen and fulvestrant on endogenous miR-221 (left panel) and miR-222 (right panel) expression. MCF7 cells were serum/E2 starved for 3 days and then treated with 10 nM 17β-estradiol (E2), 1 μM 4-hydroxytamoxifen (OHT), 100 nM fulvestrant alone or in the specified combinations, and cells were collected at the indicated time points. qRT–PCR was performed to determine miR-221/222 expression, relative to those in dimethyl sulfoxide (DMSO)-treated cells (mean±s.e., n = 3). **P<0.01.

Figure 2

Ectopic expression of miR-221/222 increases ER-independent growth and confers resistance to fulvestrant. (a) Ectopic expression of miR-221/222 in MCF7 cells. MCF7 cells were transiently transfected with 50 nM pre-miRNA precursors. Following a 72-h incubation, cells were subjected to qRT–PCR (upper panel) and immunoblotting with anti-ERα, p27^Kip1 and GAPDH antibodies (lower panel). The miR-221/222 levels in scramble control-treated MCF7 cells were normalized as 1 (mean±s.e., n = 3). **P<0.01. (b) ER-independent growth rate of pre-miRNA-transfected cells (MCF7, MCF7-T and BT474). Cells were seeded in E2-free media one day before transfection. MTT assay was performed to determine cell numbers at the indicated time points (absorbance at 600 nM linearly correlated with cell number; mean±s.e., n = 6) **P<0.01. (c) Cell morphology changes in pre-miRNA-transfected MCF7 cells. One day after E2 starvation, MCF7 cells were transfected with pre-221/222 and further cultured in E2-free media for 3 days, followed by phase-contrast microphotography (magnification ×20 objective) and fluorescence microscopy (magnification ×60 oil immersion objective. Bar, 10 μM). (d) Fulvestrant sensitivity of pre-miRNA-transfected cells (MCF7, MCF7-T and BT474). Cells were maintained in E2-free medium containing indicated doses of fulvestrant for 7 days. Relative cell growth rates (drug versus vehicle) were determined by MTT assay (mean±s.e., n = 6). **P<0.01.

Figure 3

miR-221/222 knockdown inhibits cell proliferation in fulvestrant-resistant MCF7-F cells. (a) Knockdown of miR-221/222 in MCF7-F cells. MCF7-F cells were transiently cotransfected with 100 nM of 2′-O-Me-antagomirs (si-221 and/or si-222). Following a 24, 48 or 72-h incubation, qRT–PCR (upper panel) and immunoblotting (lower panel) with anti-p27^Kip1 and GAPDH antibodies were performed. The miR-221/222 levels in negative control-treated MCF7-F cells were normalized as 1 (mean±s.e., n = 3). **P<0.01. (b) Inhibited proliferation of MCF7-F cells after knockdown of miR-221 and/or miR-222. The growth rate was determined using BrdU incorporation assay (mean±s.e., n = 6). **P<0.01. (c) Clonogenic activity of MCF7-F cells. Sorted antagomir-treated MCF7-F cells were cultured in growth medium for 2 weeks and colonies containing >50 cells were scored (mean±s.e., n = 3). *P<0.05.

Figure 4

miR-221/222-regulated genes and signaling pathways. (a) Left panel: Two-dimensional hierarchical clustering of genes differentially expressed in si-221 or si-222-transfected MCF7-F cells compared with negative control-transfected MCF7-F cells. Each row represents a single gene. Red, genes with higher expression levels; green, genes with lower expression levels. Right panel: representative-signaling pathways significantly regulated by miR-221 or -222-regulated genes. Impact factor strength is shown. (b) The same analysis was performed for pre-221 or pre-222-transfected MCF7 cells.

Figure 5

Time-course gene expression after ectopic expression of miR-221/222 in MCF7 cells. (a) qRT–PCR results showing genes with altered expression within 12 h after transfection of pre-miR-221/222 (solid lines), compared with scramble control (dash lines, normalized to 1). (b) (c) Genes with altered expression within 24 h or 48 h after transfection (mean±s.e., n = 2, with two independent experiments). *P<0.05, **P<0.01.

Figure 6

miR-221/222 upregulates and activates β-catenin. (a) Upregulated β-catenin mRNA in MCF7-T and BT474 cells. qRT–CR results indicated the time-course changes of β-catenin mRNA in MCF7-T cells (right panel) and the β-catenin mRNA level 48 h after transfection in BT474 cells (left panel) (mean±s.e., n = 2, with two independent experiments). **P<0.01. (b) Increased β-catenin protein level in the nuclear fraction (MCF7, MCF7-T and BT474). Immunoblotting results showed the protein level of β-catenin in nucleus and cytoplasm, whereas TATA binding protein (TBP) was used as loading control for nuclear proteins (upper panel) and β-tubulin as the control for cytoplasmic proteins (lower panel). (c) Increased β-catenin transcriptional activity. MCF7 cells were transfected with TOPflash or FOPflash reporter plasmids, together with β-gal pasmid for 48 h. Luciferase activities were measured using a luminometer, and β-gal assays were performed to normalize transfection efficiencies. (mean±s.e., n = 3). *P<0.05. (d) Inhibition of β-catenin activity prevents cell growth of pre-miRNA-treated MCF7 cells. Cells were grown in phenol red-free medium with 5% csFBS in the presence of doses of epigallocatechin-3-gallate (EGCG) for 6 days and cell survival rates were determined by MTT assay. (mean±s.e., n = 6). *P<0.05, **P<0.01.

Figure 7

Overexpression of β-catenin partially confers MCF7 cells resistance to fulvestrant. (a) Decreased fulvestrant sensitivity of β-catenin-overexpressing MCF7 cells. Cells were cultured in phenol red-free medium with 10% csFBS and MTT assay was performed 6 days after adding fulvestrant. (mean±s.e., n = 6). **P<0.01. (b) Immunoblotting results indicating the protein level of β-catenin.

Figure 8

miR-221/222 alter cell sensitivity to TGF-β1-induced growth inhibition. (a) TGF-β1 sensitivity of MCF7 cells. Cells were treated with doses of TGF-β1 for 4 days and cell growth rates (drug versus vehicle) were determined by MTT assay. (mean±s.e., n = 6). *P<0.05, **P<0.01. (b) TGF-β1 sensitivity of MCF7-F cells determined as in (a).