MicroRNA-221/222 confers tamoxifen resistance in breast cancer by targeting p27(kip1).

Abstract

Abstract Abstract #3023 Background: Breast cancer is the most common malignancy in women, accounting for 31% of all female cancers. Over two-thirds of breast cancers exhibit high concentrations of estrogen receptor, which contribute to tumor growth and progression. Blocking the steroid hormone pathway with tamoxifen and/or oophorectomy has been shown to be effective in this patient population. However, approximately 30% of the breast cancer is resistant to tamoxifen. Recent studies have highlighted the key regulatory roles of microRNAs (miR) in all fundamental cellular processes in animals and plants including primary human cancers. We hypothesized that alteration in the expression of specific miRs in breast cancer could contribute to tamoxifen resistance.
 Methods: To test this hypothesis, we performed microRNA microarray analysis using MCF-7 cell lines that are either sensitive (parental) or resistant to tamoxifen (4-hydroxy tamoxifen resistant-OHTR). Using Real-time RT-PCR we validated altered expression of the miRs in both the cell culture model and the primary human breast cancer tissues. Cells overexpressing miR-221/222 or p27(kip1) were created by transfection of mammalian expression vectors using Lipofectamine2000 followed by G418 selection. Cell viability upon tamoxifen treatment was measured by MTT assay, extent of apoptosis was monitored by Western blot analysis of PAPR and Caspase cleavage and cell cycle profile was studied using Flow cytometry.
 Results: Eight miRs were found to be significantly upregulated while seven miRs were significantly down-regulated in the OHTR cells compared to parental MCF-7 cells. The increased expression of three upregulated (miR-221, miR-222 and miR-181) and three downregulated (miR-21, miR-342 and miR-489) miRs was later validated in the cell lines by real-time RT-PCR. In addition, the level of miR-221 and miR-222 was significantly elevated in Her2/neu(+) primary human breast cancer tissues, compared to Her2/neu(-) tissue samples. The Her2/neu expressing tumors are known to be relatively resistant to endocrine therapy. Ectopic expression of miR-221/miR-222 in parental MCF-7 cells rendered the cells more tolerant to tamoxifen than the control cells. The cell cycle inhibitor p27/Kip1, a known target of miR-221/miR-222 was significantly reduced in both OHTR cells and miR-221/222 overexpressing MCF-7 cells, which was consistent with the upregulation of the miRs. Ectopic expression of p27/Kip1 in the resistant OHTR cells enhanced cell death when exposed to tamoxifen.
 Conclusion: This study has revealed a specific miR signature for the tamoxifen-resistant breast cancer and an important cell cycle inhibitor target of the altered miR, which could be used as a prognostic marker for the drug-resistant breast cancer. Further studies of other miRs differentially expressed in tamoxifen resistant cell lines, will help us not only in identifying such patients but also may serve as a therapeutic target in the future. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3023.