MicroRNA-221/222 negatively regulates estrogen receptor alpha and is associated with tamoxifen resistance in breast cancer

Abstract

A search for regulators of estrogen receptor alpha (ERalpha) expression has yielded a set of microRNAs (miRNAs) for which expression is specifically elevated in ERalpha-negative breast cancer. Here we show distinct expression of a panel of miRNAs between ERalpha-positive and ERalpha-negative breast cancer cell lines and primary tumors. Of the elevated miRNAs in ERalpha-negative cells, miR-221 and miR-222 directly interact with the 3'-untranslated region of ERalpha. Ectopic expression of miR-221 and miR-222 in MCF-7 and T47D cells resulted in a decrease in expression of ERalpha protein but not mRNA, whereas knockdown of miR-221 and miR-222 partially restored ERalpha in ERalpha protein-negative/mRNA-positive cells. Notably, miR-221- and/or miR-222-transfected MCF-7 and T47D cells became resistant to tamoxifen compared with vector-treated cells. Furthermore, knockdown of miR-221 and/or miR-222 sensitized MDA-MB-468 cells to tamoxifen-induced cell growth arrest and apoptosis. These findings indicate that miR-221 and miR-222 play a significant role in the regulation of ERalpha expression at the protein level and could be potential targets for restoring ERalpha expression and responding to antiestrogen therapy in a subset of breast cancers.

FIGURE 1.

Frequently increased expression of miR-221 and miR-222 in ERα-negative breast cancer. A, partial heat map of miRNA microarray analysis of ERα-positive versus ERα-negative breast cancer cell lines and primary tumors. Several miRNAs were significantly elevated in ERα-negative cells. B and C, elevated levels of miR-221 and miR-222 in ERα-negative breast cancer cell lines and primary tumors. Total RNAs from the cell lines and primary tumors were subjected to Northern blot (B) and quantitative RT-PCR (C) analyses. U6 small nuclear RNA (snRNA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as loading controls. The blots were quantified by dividing miR-221 and miR-222 signals by U6 and by dividing ERα by glyceraldehyde-3-phosphate dehydrogenase. ERα-negative tumors overexpressing both miR-221 and miR-222 are labeled by asterisks, and the tumors also expressing ERα mRNA are indicated by triangles (C). M stands for marker. D, representation of the inverse correlation of expression of ERα and miR-221/miR-222. Breast tumor specimens were immunohistochemically stained with anti-ERα antibody (first and third panels). The second and fourth panels are the same specimens that were hybridized with the LNA-miR-221 and LNA-miR-222 probes using miRNA locked nucleic acid in situ hybridization as described under “Experimental Procedures.”

FIGURE 2.

miR-221 and miR-222 negatively regulate ERα and render cells resistant to tamoxifen. A, sequence alignment of the human miR-221 and miR-222 seed sequences with two regions of the ERα 3′-UTR. One region is conserved (ERα1; upper), and the other is not (ERα 2; lower). Mutants of pmiR-ERα1-5 (seed sequence mutation) and pmiR-ERα1-3 are shown (middle), and the mutant nucleotides are labeled in red. B, miR-221 and miR-222 inhibit ERα expression in a dose-dependent manner. MCF-7 cells were transiently transfected with indicated amounts of BLOCK-iT-miR-221 and BLOCK-iT-miR-222 expression plasmids. Following a 72-h incubation, cells were subjected to immunoblotting with anti-ERα (first panel) and β-actin (second panel) antibodies and quantitative RT-PCR analysis (third through fifth panels). Expression of transfected miR-221 and miR-222 is shown in the third and fourth panels. U6 small nuclear RNA (snRNA) is a loading control. Quantification was done by dividing ERα signals by actin. M stands for marker. C, decrease in ERα protein but not mRNA levels by stable expression of miR-221 or miR-222 in MCF-7 and T47D cells. Following transfection of BLOCK-iT-miR-221 or BLOCK-iT-miR-222, cells were selected with blasticidin. Stably transfected cells were subjected to quantitative RT-PCR (upper panels), Western blot (middle panels), and RT-PCR (lower panels) analyses. Dividing ERα signals by actin (Western) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; RT-PCR) was used to quantify the protein and mRNA levels of ERα, respectively. D, immunofluorescence staining of parental MCF-7 cells (panels A-D) and cells transiently transfected with the GFP vector (panels E-H), GFP-miR-221 (panels I-L), or GFP-miR-222 (panels M-P) with anti-ERα antibody (panels C, G, K, and O). Cells transfected with vector (e.g. expressing only GFP) exhibited the same levels of ERα as did parental cells (panels A-D). ERα signals in miR-221- and miR-222-transfected cells (arrowheads) were significantly lower than in untransfected surrounding cells (arrows). Quantitation of ERα-positive cells is shown in the bar graph. DAPI, 4′,6-diamidino-2-phenylindole. E, ectopic expression of miR-221 and miR-222 reduces tamoxifen-induced cell death. MCF-7 and T47D cells were stably transfected with miR-221, miR-222, and the vector alone as described for C. Following treatment with or without tamoxifen, cell viability was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The experiment was repeated three times in triplicate. Asterisks indicate p < 0.05. The gels show expression of transfected miR-221 and miR-222.

FIGURE 3.

miR-221 and miR-222 interact with the conserved site of the ERα 3′-UTR. A, pmiR-ERα1 but not pmiR-ERα2 reporter activity is reduced only in miR-221/222-positive MDA-MB-468 cells. The pmiR-ERα1-Luc and pmiR-ERα2-Luc plasmids were introduced into MCF-7 and MDA-MB-468 cells together with β-galactosidase. Luciferase activity was measured and normalized after a 36-h incubation. B, ectopic expression of miR-221 or miR-222 inhibits pmiR-ERα1-Luc and pmiR-ERα1-3-Luc but not seed sequence mutant pmiR-ERα1-5-Luc. MCF-7 cells were transfected with the indicated plasmids and assayed for luciferase activity after a 36-h incubation. The inset shows expression of transfected miR-221 and miR-222. M stands for marker. snRNA, small nuclear RNA. C and D, the activities of pmiR-ERα1-Luc and pmiR-ERα1-3-Luc but not seed sequence mutant pmiR-ERα1-5-Luc are reduced in MDA-MB-468 cells (C), which are partially abrogated by knockdown of miR-221/222 (D). MDA-MB-468 cells were transfected with the indicated plasmids and 2′-O-Me. Following a 36-h incubation, luciferase reporter assay was performed as described under “Experimental Procedures.” All experiments were repeated three times in triplicate. Expression of miR-221 and miR-222 is shown in the inset.

FIGURE 4.

Knockdown of miR-221 and/or miR-222 partially restores ERα expression and tamoxifen sensitivity in ERα protein-negative/mRNA-positive cells. A, expression of ERα protein and mRNA in breast cancer cell lines. Four ERα-positive and eight ERα-negative cell lines were subjected to RT-PCR (upper panels) and Western blot (lower panels) analyses for ERα expression. B and C, partial restoration of ERα expression by knockdown of miR-221 and miR-222. MDA-MB-468 or MCF-10A cells were transfected with 2′-O-Me-anta-miR-221 and/or 2′-O-Me-anta-miR-222. After a 72-h incubation, cells were subjected to Northern (B) and Western (C) blot analyses with the indicated probes and antibodies, respectively. Quantification was performed as described for Figs. 1 and 2. D and E, knockdown of miR-221 and miR-222 sensitizes MDA-MB-468 cells to tamoxifen-induced cell death. 2′-O-Me-anta-miR-221- and/or 2′-O-Me-anta-miR-222-transfected MDA-MB-468 cells from B were treated with the indicated doses of tamoxifen (TAM). Cell survival and apoptosis were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (D) and using a Cell Death Detection ELISAPLUS kit (E). Each experiment was repeated three times in triplicate. Asterisks represent p < 0.05 of tamoxifen-induced cell death between miR-221- and/or miR-222-knocked down cells and scrambled 2′-O-Me-treated cells.