MicroRNA-221/222 confers tamoxifen resistance in breast cancer by targeting p27Kip1

Abstract

We explored the role of microRNAs (miRNAs) in acquiring resistance to tamoxifen, a drug successfully used to treat women with estrogen receptor-positive breast cancer. miRNA microarray analysis of MCF-7 cell lines that are either sensitive (parental) or resistant (4-hydroxytamoxifen-resistant (OHT(R))) to tamoxifen showed significant (>1.8-fold) up-regulation of eight miRNAs and marked down-regulation (>50%) of seven miRNAs in OHT(R) cells compared with parental MCF-7 cells. Increased expression of three of the most promising up-regulated (miR-221, miR-222, and miR-181) and down-regulated (miR-21, miR-342, and miR-489) miRNAs was validated by real-time reverse transcription-PCR. The expression of miR-221 and miR-222 was also significantly (2-fold) elevated in HER2/neu-positive primary human breast cancer tissues that are known to be resistant to endocrine therapy compared with HER2/neu-negative tissue samples. Ectopic expression of miR-221/222 rendered the parental MCF-7 cells resistant to tamoxifen. The protein level of the cell cycle inhibitor p27(Kip1), a known target of miR-221/222, was reduced by 50% in OHT(R) cells and by 28-50% in miR-221/222-overexpressing MCF-7 cells. Furthermore, overexpression of p27(Kip1) in the resistant OHT(R) cells caused enhanced cell death when exposed to tamoxifen. This is the first study demonstrating a relationship between miR-221/222 expression and HER2/neu overexpression in primary breast tumors that are generally resistant to tamoxifen therapy. This finding also provides the rationale for the application of altered expression of specific miRNAs as a predictive tamoxifen-resistant breast cancer marker.

FIGURE 1.

miRNA expression profile of tamoxifen-sensitive and tamoxifen-resistant MCF-7 cells. A, shown are the results from cluster analysis. Total RNAs isolated from three biological replicates of tamoxifen-sensitive MCF-7 cells and tamoxifen-resistant MCF-7 cells (OHT^R) were subjected to miRNA microarray analysis. miRNA expression data were normalized to the average median of all the genes present in the array. miRNAs expressed at least 1.5-fold higher or 50% lower in OHT^R cells compared with MCF-7 cells were considered for cluster analysis. B, shown are the miRNAs that are up-regulated and down-regulated in OHT^R cells. C, validation is shown. Total RNAs isolated from three biological replicates of MCF-7 and OHT^R cells were subjected to real-time RT-PCR to validate differential expression of miR-221, miR-222, miR-181b, miR-21, miR-342, and miR-489. Each assay was done in triplicate, and expression of miRNA was normalized to snRNA RNU6B. D, the expression of miR-221 and miR-222 was analyzed in HER2/neu-positive and HER2/neu-negative primary human breast cancer tissues. Total RNAs isolated from formalin-fixed paraffin-embedded tissue sections were subjected to real-time RT-PCR with specific miRNA assay kits. Expression of snRNA RNU6B was used as normalizer. Normalized expression of the miRNAs was compared between the HER2/neu-positive (HER2/Neu +ve) and HER2/neu-negative (HER2/Neu -ve) samples using a Whisker plot. The asterisk indicate the outliers. E, total RNAs isolated from MCF-7 and ZR75.1 cells were subjected to real-time RT-PCR to analyze the expression levels of miR-221, miR-222, and HER2. miRNA levels were normalized to snRNA RNU6B, and HER2 levels were normalized to 18 S rRNA.

FIGURE 2.

Ectopic expression of miR-221/222 in MCF-7 cells results in increased tamoxifen resistance. A, expression of miR-221 and miR-222 was analyzed using total RNA isolated from miR-221/222-transfected MCF-7 cells by real-time RT-PCR and normalized to snRNA RNU6B. -Fold increase is shown below the bars. B, the G418-selected pool of miR-221/222-expressing MCF-7 cells, G418-selected clone 9 (MCF-7/221/222), and the empty vector-transfected MCF-7 cell pool were treated with 5 μm tamoxifen for 72 h. Cell metabolic activity in the presence of the drug was measured every 24 h using the MTT assay. The metabolic activity of cells at 0 h was taken as 1. The results are the means ± S.D. of triplicate assays. C, vector- and miR-221/222-expressing MCF-7 cells were treated with 0, 15, and 20 μm tamoxifen for 16 h. The cells were photographed using a phase-contrast microscope. O/E, overexpressing. D, whole cell extracts from the vector- and miR-221/222-transfected MCF-7 cells were separated by SDS-PAGE and probed with antibodies against PARP and caspase-7 that detect respective intact and cleaved products. The blot was reprobed with anti-Ku-70 antibody to ensure equal protein loading. The signal in each band was quantified using Kodak Imaging software. Quantification of both caspase-7 and PARP (uncleaved and cleaved) was plotted after normalization to Ku-70. The results are representative of two independent experiments. E, the vector- and miR-221/222-expressing MCF-7 cells were treated with 20 μm tamoxifen (Tam) for 24 h. The cells were fixed and stained with propidium iodide, and cell cycle distribution was monitored by flow cytometry in a FACSCalibur. The percentage of apoptotic cells (sub-G₁ peak) in vector (Vec)- and miR-221/222-expressing cells is represented in the bar diagram. Similar results were obtained from three independent experiments.

FIGURE 3.

p27^Kip1, a target of miR-221/222, imparts tamoxifen sensitivity to MCF-7 cells. A, whole cell extracts from MCF-7 cells, OHT^R cells, and miR-221/222-transfected and vector (Vec)-transfected MCF-7 cells were subjected to SDS-PAGE and probed with anti-p27^Kip1 antibody. The membrane was reprobed with anti-Ku-70 antibody, and p27^Kip1 expression was normalized to Ku-70 protein. O/E, overexpressing. B, total RNAs from MCF-7 cells, OHT^R cells, and miR-221/222-transfected and vector-transfected MCF-7 cells were analyzed by real-time RT-PCR with primers specific for p27^Kip1 and 18 S rRNA. C, whole cell extract from the p27^Kip1-transfected OHT^R cell pool was subjected to Western blot analysis with anti-p27^Kip1 antibody and reprobed with anti-Ku-70 antibody. D, OHT^R cells transfected with empty vector and p27 expression vector were treated with 5 μm tamoxifen for 72 h. Cell metabolic activity was measured every 24 h using the MTT assay. The metabolic activity of cells at 0 h was taken as 1. The results are the means ± S.D. of triplicate assays. E, vector-overexpressing (panels A–C) and p27^Kip1-overexpressing (D–F) OHT^R cells were treated with 0 and 15 μm tamoxifen for 16 h. The cells were photographed using a phase-contrast microscope. The small arrows in panels G and H indicate autophagosome-like bodies. F, whole cell extracts from the vector-transfected and p27^Kip1-transfected OHT^R cells were subjected to SDS-PAGE and probed with antibodies against caspase-7 and PARP. The blot was reprobed with anti-Ku-70 antibody to normalize protein loading. The signal in each band was quantified using Kodak Imaging software. Quantification of uncleaved and cleaved caspase-7 and PARP is presented in the bar diagrams. The results are representative of three independent experiments. TAM, tamoxifen.