Oncogene (2011) 30, 1082–1097 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE MicroRNA-221/222 confers breast cancer fulvestrant resistance by regulating multiple signaling pathways

X Rao1, G Di Leva2,MLi3, F Fang3, C Devlin4,6, C Hartman-Frey3, ME Burow5, M Ivan4,6, CM Croce2 and KP Nephew1,3,6

1Interdisciplinary Biochemistry Graduate Program, Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN, USA; 2Department of Molecular Virology, Immunology and Medical Genetics, Comprehensive Cancer Center, Ohio State University, Columbus, OH, USA; 3Department of Medical Sciences, Indiana University School of Medicine, Bloomington, IN, USA; 4Department of Medicine, Department of Immunology and Microbiology, Indiana University School of Medicine, Indianapolis, IN, USA; 5Department of Medicine, Tulane University School of Medicine, New Orleans, LA, USA and 6IU Simon Cancer Center and Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, IN, USA

Fulvestrant is a selective estrogen downregulator Keywords: microRNAs; breast cancer; antiestrogen; (SERD) and highly effective antagonist to hormone-sensitive drug resistance; fulvestrant 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 Introduction aberrantly expressed microRNAs (miRNAs) in acquired fulvestrant resistance, we compared antiestrogen-resistant -a (ERa), the primary mediator of and -sensitive breast cancer cells, revealing the over- estrogen action, plays a pivotal role in the development expression of miR-221/222 in the SERD-resistant cell and progression of breast cancer (Ariazi et al., 2006). lines. Fulvestrant treatment of estradiol (E2)- and fulvestrant- As over 70% of breast cancers overexpress ERa, sensitive MCF7 cells resulted in increased expression of endo- targeting the receptor by endocrine therapy is consid- genous miR-221/222. Ectopic upregulation of miR-221/222 ered the most important treatment for patients with ER- in estrogen receptor-a (ERa)-positive cell lines counteracted positive breast tumors (Ariazi et al., 2006). Tamoxifen, a the effects of E2 depletion or fulvestrant-induced cell death, selective estrogen receptor modulator (Jordan, 2003), thus also conferring hormone-independent growth and and aromatase inhibitors (AIs), which block estrogen fulvestrant resistance. In cells with acquired resistance to synthesis (Smith and Dowsett, 2003), are effective first- fulvestrant, miR-221/222 expression was essential for cell line endocrine therapies that significantly improve relapse- growth and cell cycle progression. To identify possible free and overall survival of all stages of ER-positive breast miR-221/222 targets, miR-221- or miR-222- induced cancer (Nicholson and Johnston, 2005). However, acquired alterations in global expression profiles and target gene resistance to both tamoxifen and AI typically develops expression at distinct time points were determined, revealing after prolonged treatment in a majority of initially that miR-221/222 overexpression resulted in deregulation of responsive breast cancers (Normanno et al., 2005), and multiple oncogenic signaling pathways previously associated nearly 50% of the advanced ER-positive breast cancer with drug resistance. Activation of b-catenin by miR-221/222 patients do not respond to tamoxifen or AI in the first- contributed to estrogen-independent growth and fulvestrant line setting (Normanno et al., 2005). resistance, whereas TGF-b-mediated growth inhibition was Resistance to AIs and tamoxifen is associated with repressed by the two miRNAs. This first in-depth investiga- activation of several growth factor signaling pathways, tion into the role of miR-221/222 in acquired fulvestrant including human epidermal growth factor receptor 2 resistance, a clinically important problem, demonstrates that (HER2) and insulin-like growth factor I receptor, which these two ‘oncomirs’ may represent promising therapeutic ‘cross-talk’ with the ERa signaling pathway, resulting in targets for treating hormone-independent, SERD-resistant an activation of various mitogen-activated breast cancer. kinases (MAPKs) and PI3K/AKT involved in cell Oncogene (2011) 30, 1082–1097; doi:10.1038/onc.2010.487; survival and proliferation (Ali and Coombes, 2002; published online 8 November 2010 Nicholson and Johnston, 2005; Normanno et al., 2005). As the transcriptional program of ERa continues to play a critical role in these acquired resistance mechan- Correspondence: Professor KP Nephew, Medical Sciences Program, isms, second-line endocrine therapies, using alternative Indiana University School of Medicine, Jordan Hall 302, 1001 E. ERa-inhibitory mechanisms, have been developed Third Street, Bloomington, IN 47405-4401, USA. (McDonnell, 2006). One of these, the pure steroidal E-mail: [email protected] Received 19 January 2010; revised 21 August 2010; accepted 10 antiestrogen fulvestrant (Faslodex, ICI 182,780, Astra- September 2010; published online 8 November 2010 Zeneca Corp., London, UK), completely suppresses ERa MicroRNA-221/222 and fulvestrant-resistant breast cancer X Rao et al 1083 activity, inactivating both ERa-mediated genomic and with multiple (ER-independent) growth-promoting, non-genomic signaling, and is now approved for the oncogenic pathways are critical for acquired selective treatment of postmenopausal women following failure estrogen receptor downregulator (SERD) resistance. We of previous antiestrogen therapy (tamoxifen and AIs) further validated that miR-221/222 activated b-catenin (Howell, 2006a). However, despite its potent antitumor signaling and repressed TGF-b-signaling-induced effects, fulvestrant does not circumvent the development growth inhibition. These results indicate vital, multi- of antiestrogen resistance (Howell, 2006b). We (Fan et al., functional roles of miR-221/222 in the acquisition of 2006) and others (Nicholson et al., 2005; Normanno et al., fulvestrant-resistant breast cancer. 2005) have shown that acquired resistance to fulves- trant is an ERa-independent phenomenon, involving constitutive activation of autocrine-regulated growth- Results stimulatory pathways, ultimately dissociating breast cancer cells from ERa-mediated growth, although the miR-221/222 expression is increased in antiestrogen- mechanism(s) of upregulation of these mitogenic path- resistant breast cancer cell lines and is upregulated ways remains unknown. by fulvestrant in antiestrogen-sensitive MCF7 cells MicroRNAs (miRNAs) are an abundant class of To investigate the association between miR-221/222 endogenous small non-coding RNAs (20–22 nucleotides expression and antiestrogen resistance, four different in length) that regulate crucial biological processes, antiestrogen-resistant human breast cancer cell lines including development, differentiation, apoptosis and were compared, each derived by different mechanisms. proliferation (Bushati and Cohen, 2007). Imperfect Tamoxifen-resistant (MCF7-T) and fulvestrant-resistant pairing of miRNAs with the 30 untranslated region of (MCF7-F) MCF7 sublines were established by pro- target mRNAs generally results in their destabilization longed growth of MCF7 cells in tamoxifen- or or ribosomal blockade (Bushati and Cohen, 2007). fulvestrant-containing media, respectively (Fan et al., Deregulation of miRNAs is now considered a hallmark 2006). MCF7-T cells remained responsive to fulvestrant of cancer (Calin and Croce, 2006), in which they can but with reduced drug sensitivity compared with the function as oncogenes or tumor suppressors (Garzon parental MCF7 cells, whereas MCF7-F cells were et al., 2009). In breast cancer, miR-125b, -145, -21 and fulvestrant-resistant and also cross-resistant to tamox- -155 have been shown the most significantly deregulated ifen. The expression profile analysis indicated that miRNAs (Iorio et al., 2005) and miR-7, -128a, -210, and subsets of clinically relevant (prognostic markers, -516-3p have been strongly associated with cancer clinical outcome and ERa signature genes) are altered in progression (Foekens et al., 2008). Furthermore, miR- these two cell lines (Fan et al., 2006), making them 10b may play a role in breast cancer invasion and valuable models for investigating the molecular events metastasis (Ma et al., 2007), and miR-206 is inversely underlying the development of antiestrogen resistance. correlated with ERa expression in human breast tumors Tumor necrosis factor-resistant (MCF7-TNR) cells were and appears to target ERa (Adams et al., 2007; Kondo obtained by prolonged exposure of MCF7 cells to et al., 2008). Estradiol (E2)-regulated miRNA signa- increasing concentrations of tumor necrosis factor-a tures, and miRNA regulation of ERa and its down- (Weldon et al., 2004), whereas MCF7-Mek5 cells were stream transcriptional response to E2 have also recently established by stably transfecting MCF7 cells with been reported (Bhat-Nakshatri et al., 2009; Castellano constitutively active mitogen-activated protein kinase et al., 2009), and an important role for miRNAs in ERa kinase 5 (MEK5) construct (Zhou et al., 2008). Both the biology is beginning to emerge (Klinge, 2009). MCF7-TNR and -Mek5 cell lines were resistant to tumor Although altered expression of miRNAs in breast necrosis factor-a- (Zhou et al., 2008) and fulvestrant- cancer has been widely reported (Iorio et al., 2005, 2008; induced apoptosis (unpublished data). Real-time qRT– Heneghan et al., 2009), the functional consequences of PCR results revealed upregulation of miR-221/222 in aberrant expression of specific miRNAs in antiestrogen- MCF7-T cells (Figure 1a), consistent with a previous resistant disease remain unclear. Overexpression of study (Miller et al., 2008). However, in the fulvestrant- miR-221 and miR-222 has been observed in a number resistant cells (MCF7-F, MCF7-Mek5 and MCF7- of advanced malignancies (Galardi et al., 2007; Gillies TNR), miR-221/222 were markedly overexpressed and Lorimer, 2007; Visone et al., 2007; Felicetti et al., (Po0.01, versus MCF7-T) (Figure 1a), suggesting a 2008; Fornari et al., 2008; Garofalo et al., 2009; Pineau stronger association of miR-221/222 with pure anti- et al., 2010), including ERa-negative primary tumors estrogen resistance. (Zhao et al., 2008), and has also been associated with Considering the different mechanisms of tamoxifen tamoxifen resistance (Miller et al., 2008). Our previous and fulvestrant action, we next examined the effects of miRNA microarray data also indicated miR-221/222 these two drugs on endogenous miR-221/222 expres- upregulation in acquired fulvestrant resistance (Xin sion. To avoid the effects of medium-derived estrogen, et al., 2009). In the current study, we used antiestro- MCF7 cells were cultured in estrogen-free medium for 3 gen-sensitive and -resistant breast cancer cell line models days and then treated with different drug combinations. to comprehensively investigate the role of miR-221/222 Although expression of miR-221/222 was not signifi- in hormone-independent growth and acquired resistance cantly altered by 17b-estradiol (E2), 4-hydroxytamoxifen to fulvestrant. miR-221/222-transfected cells demon- (OHT) or E2 combined with OHT during the first 24 h, strated that global changes associated these drug treatments actually repressed (Po0.01) miR-

Oncogene MicroRNA-221/222 and fulvestrant-resistant breast cancer X Rao et al 1084 221/222 expression at subsequent time points (48–96 h; investigated the role of miR-221/222 in the acquisition Figure 1b). By contrast, fulvestrant, alone or in of fulvestrant resistance. Fulvestrant-sensitive MCF7, combination with E2, increased (Po0.01) endogenous MCF7-T and BT474 breast cancer cells were transfected miR-221/222 expression after 48-h treatment with pre-miRNA to ectopically overexpress miR-221/ (Figure 1b). As miR-221/222 were overexpressed in 222, as confirmed by qRT–PCR and immunoblotting fulvestrant-resistant cell lines (Figure 1a), these results analysis for suppressed p27Kip1 and ERa protein levels, suggest that fulvestrant treatment (or removal of ERa) two well-known targets of these miRNAs (Galardi et al., might initially induce miR-221/222 expression during 2007; Zhao et al., 2008) (Figure 2a). Although the the development of SERD resistance. growth of MCF7 cells was retarded in estrogen-free medium, the proliferation of miR-221 and/or miR-222- miR-221/222 confer estrogen-independent growth overexpressing MCF7 cells was significantly (Po0.01) and fulvestrant resistance accelerated (Figure 2b), which was further confirmed As miR-221/222 expression was induced by fulvestrant using MCF7-T and BT474 cell lines (Figure 2b). After and associated with fulvestrant resistance, we next E2 depletion for four days, the scramble control siRNA-

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 ¼ 3). **Po0.01. The following t-test results were obtained: MCF7-F, MCF7-Mek5 or MCF7-TNR versus MCF7-T, Po0.002. (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 17b-estradiol (E2), 1 mM 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 ¼ 3). **Po0.01.

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-ERa, p27Kip1 and GAPDH antibodies (lower panel). The miR-221/222 levels in scramble control-treated MCF7 cells were normalized as 1 (mean±s.e., n ¼ 3). **Po0.01. (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 ¼ 6) **Po0.01. (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 mM). (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 ¼ 6). **Po0.01.

Oncogene MicroRNA-221/222 and fulvestrant-resistant breast cancer X Rao et al 1085 treated MCF7 cells exhibited previously described of autophagy) antibody revealed punctated patterns of morphological signs of cellular damage (Gozuacik and LC3 immunofluorescence in scramble control-treated Kimchi, 2004; Miller et al., 2008), which were not MCF7 cells, whereas LC3 immunofluorescence in miR- observed in miR-221 and/or miR-222-overexpressing 221 and/or miR-222-overexpressing cells was diffused MCF7 cells (Figure 2c, upper panel, arrows indicated). (Figure 2c, lower panel). In addition, MCF7 cells Immunofluorescence staining with anti-LC3B (a marker overexpressing miR-221 and/or miR-222 displayed a

Oncogene MicroRNA-221/222 and fulvestrant-resistant breast cancer X Rao et al 1086 rounded phenotype and were loosely attached to the Knockdown of miR-221/222 in MCF7-F cells inhibits culture surface (Figure 2c), morphologic characteristics cell proliferation of the aggressive MCF7-F cells (Fan et al., 2006). To further investigate the role of miR-221/222 in To further examine miR-221/222 effects on fulves- fulvestrant resistance, MCF7-F cells were transfected trant sensitivity, hormone-sensitive MCF7 cells were with 20-O-Me-221 (si-221) and 20-O-Me-222 (si-222), or treated with different doses of fulvestrant for 7 days, control 20-O-Me-eGFP (NEG) antagomirs. After 24 h, showing strong growth inhibition in the control- miR-221 and miR-222 expression was dramatically transfected cells (Figure 2d, left panel); however, reduced in the si-221/222-transfected MCF7-F cells, that growth inhibition was markedly blunted in and p27Kip1 protein levels were restored (Figure 3a; the miR-221 and/or miR-222-overexpressing cells Supplementary Figure S3). BrdU cell proliferation (Figure 2d, Supplementary Figure S1). Ectopically assays indicated that si-221 or si-222 significantly overexpressing miR-221/222 in MCF7-T cells (which decreased MCF7-F cell growth by decreasing the cell have a slightly higher miR-221/222 expression than population in S-phase, with si-222 demonstrating MCF7 (Figure 1a) but are still sensitive to fulvestrant) stronger inhibitory activity (Figure 3b), perhaps due to also significantly decreased fulvestrant sensitivity cross-inhibition of miR-221 by si-222 (Supplementary (Figure 2d middle panel). Similar results were observed Figure S4). Cotransfection of si-221 and si-222 resulted in BT474 cells, which have low miR-221/222 expression in further inhibition of cell proliferation (Figure 3b). (data not shown) but are less fulvestrant sensitive than As clonogenic activity of MCF7-F cells was greater than MCF7 cells (Figure 2d right panel). Immunoblotting the parental MCF7 cells (Supplementary Figure S5), we analysis (Figure 2a and Supplementary Figure S2) then examined the role of miR-221/222 in the increased further suggested that targeting p27Kip1 may be one of clonogenicity. MCF7-F cells were cotransfected with anta- the mechanisms by which miR-221/222 confers the ERa- gomirs and a Block-iT Fluorescence Oligo (Invitrogen independent, fulvestrant-resistant phenotype. Taken to- Corp., Carlsbad, CA, USA) for sorting transfected cells gether, these results indicate that miR-221/222 over- for subsequent clonogenicity assays. As shown in Figure 3c, expression in ERa-positive cell lines counteracts the si-221/222 significantly decreased the clonogenic activity effects of E2 depletion or fulvestrant-induced cell death, of MCF7-F cells. Collectively, these studies demonstrate thus also conferring hormone-independent growth and that miR-221/222 play a key role in supporting the fulvestrant resistance. proliferation of fulvestrant-resistant cells.

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 20-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-p27Kip1 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 ¼ 3). **Po0.01. (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 ¼ 6). **Po0.01. (c) Clonogenic activity of MCF7-F cells. Sorted antagomir-treated MCF7-F cells were cultured in growth medium for 2 weeks and colonies containing 450 cells were scored (mean±s.e., n ¼ 3). *Po0.05.

Oncogene MicroRNA-221/222 and fulvestrant-resistant breast cancer X Rao et al 1087 miR-221/222 regulate global gene expression and multiple Time-course gene expression after ectopic expression signaling pathways involved in acquired fulvestrant of miR-221/222 resistance As overexpressing miR-221/222 conferred estrogen- The underlying mechanism of miR-221/222-mediated independent growth and fulvestrant resistance to fulvestrant resistance appears to include downregulation MCF7 cells, we examined time-course changes in gene of the cell cycle inhibitors p27Kip1 and p57Kip2 (Galardi expression associated with this phenomenon. Based on et al., 2007; Fornari et al., 2008), in addition to ERa the above gene expression profiles, 10 genes upregulated (Zhao et al., 2008; Di Leva et al., 2010). However, recent by miR-221/222 in MCF7 cells (Supplementary Table bioinformatic analyses (John et al., 2004) predict that a S2) were chosen for further study performing qRT–PCR single miRNA is capable of regulating hundreds of at 4, 12, 24, 48, 72 and 120 h after transfection of MCF7 genes, resulting in extensive miRNA-induced mRNA cells with pre-miR-221/222. p27Kip1, a known target of degradation (Filipowicz et al., 2008), and global gene miR-221/222, decreased by 12 h after transfection expression changes (Huang et al., 2007; Nam et al., (Figure 5a). Two key components of the Wnt/b-catenin 2009). To gain further insight into the functional role of pathway, FZD5 and CTNNB1 (b-catenin), were upre- miR-221/222 in acquired fulvestrant resistance, we gulated by 12 h (Figure 5a). Increased expression of performed mRNA expression profiling after knocking TGF-b pathway components, BMPR2 and SMAD2, down miR-221 or miR-222 in MCF7-F cells. As the was observed by 24 h after transfection, whereas mRNA and protein levels of p27Kip1 (used as a control) TGFBR2 was increased by 48 h (Figures 5b and c). did not strongly correlate (Supplementary Figure S6), The cytokines CCL5 and GDF15, and cytokine we used a moderate stringency to identify significantly receptors CRCR4 were upregulated by 24 and 48 h, regulated genes (P-value o0.05 and a fold-change 41.2 respectively (Figures 5b and c). Expression of the or oÀ1.2). Two-dimensional unsupervised hierarchi- STAT1transcription factor increased by 48 h. Increased cal clustering (average linkage clustering method) expression of GADD45A, which is involved in , was utilized to visualize common expression patterns MAPK, and cell-cycle pathways was seen by 48 h between samples. Clustering results of all significantly (Figure 5c). Of the genes downregulated by miR-221/ up- or downregulated probes demonstrated a si-221 and 222, only one was decreased by 12 h after transfection si-222 cluster, and a separate control cluster (Figure 4a). (Supplementary Figure S9), suggesting that miR-221/ After downregulation of miR-221 or miR-222, respec- 222 downregulation of their targets occurs mainly at the tively, we identified 1347 or 1029 upregulated, and 912 protein level in MCF7 cells. or 909 downregulated probes, with 428 probes upregu- lated and 224 probes downregulated by both (Supple- mentary Figure S7). For in-depth analysis, we also Regulation of b-catenin expression and transcriptional included our previous gene expression profiles of pre- activity by miR-221/222 miRNA-treated MCF7 cells (Di Leva et al., 2010), and b-Catenin is a key regulator in the Wnt signal the clustering results indicated a similar pattern transduction cascade (Takahashi-Yanaga and Kahn, (Figure 4b). 2010), and we previously demonstrated a vital role of To investigate the functional role of miR-221/222- b-catenin for acquired fulvestrant resistance as upregu- regulated genes, pathway-express analysis (Draghici lation and activation of b-catenin contributed to et al., 2007) was used, and genes regulated by si-221 or estrogen-independent growth of MCF7-F (Fan et al., si-222 in MCF7-F cells were significantly enriched in 23 2006). As b-catenin mRNA levels rapidly (12 h) or 17 KEGG (Kyoto Encyclopedia of Genes and increased in MCF7 cells when overexpressing miR- Genomes) (Kanehisa and Goto, 2000) pathways (Sup- 221/222 (Figure 5a) and knockdown of miR-221 plementary Table S1), represented by p53, TGF-b, and/or miR-222 decreased b-catenin protein levels MAPK, Notch, ErbB, and Jak-STAT signaling pathway in MCF7-F cells (Supplementary Figure S10), we (Figure 4a). Genes regulated by pre-221 or pre-222 in hypothesized that miR-221/222 serves as a positive MCF7 cells were enriched in 21 or 22 KEGG pathways, regulator of b-catenin expression and transcriptional represented by phosphatidylinositol signaling system, activity. To test this hypothesis, we first confirmed MAPK, TGF-b, ErbB, p53, apoptosis and Wnt signal- the upregulation of b-catenin mRNA levels by miR- ing pathways (Figure 4b). Representative genes in these 221/222 in MCF7-T and BT474 cells (Figure 6a), pathways are listed in Table 1. Our previous study which also increased fulvestrant resistance as previ- demonstrated that both EGFR/ErbB2 and Wnt/ ously indicated (Figure 2d). As only nuclear b-catenin b-catenin pathways play a role in supporting estrogen- has been shown to be transcriptionally active (Shitashige independent cell growth of MCF7-F (Fan et al., 2006) et al., 2008), we examined nuclear b-catenin protein and the Cignal Finder Cancer Pathway Reporter Assay levels. After overexpressing miR-221 and/or miR-222, (SABiosciences, Frederick, MD, USA) also showed upregu- elevated nuclear b-catenin levels were observed in lation of Wnt signaling in MCF7-F cells compared with all three cell lines (MCF7, MCF7-T and BT474; MCF7, as well as Notch, p53, MAPK, whereas down- Figure 5b), whereas b-catenin levels in the cytoplasm regulation of TGF-b signaling (Supplementary Figure S8). were also increased in MCF7 and MCF7-T cells. These pathways are regulated by miR-221/222 (described Wnt signaling reporter analysis using TOPflash and above), further supporting the multifunctional role of FOPflash plasmids (Korinek et al., 1997) confirmed miR-221/222 in the acquisition of fulvestrant resistance. that overexpression of miR-221/222 in MCF7 cells

Oncogene MicroRNA-221/222 and fulvestrant-resistant breast cancer X Rao et al 1088

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.

Oncogene MicroRNA-221/222 and fulvestrant-resistant breast cancer X Rao et al 1089 Table 1 Cell signaling pathways altered by miR-221/222 in MCF7 and MCF7-F cells Gene symbol Genebank Fold-change Description

MCF7-F versus pre221/222 in si221/222 in MCF7 MCF7 MCF7-F

TGF-b signaling pathway TGFBR2 NM_003242 4.31 1.87 Transforming growth factor-b receptor II (70/80 kDa) BMPR2 NM_033346 3.92 1.78 À1.27 Bone morphogenetic protein receptor, type II (serine/threonine kinase) GDF15 NM_004864 4.86 À1.47 Growth differentiation factor 15 INHBA NM_002192 5.59 À1.74 Inhibin-b a (activin a, activin ab-a polypeptide) INHBC NM_005538 À2.79 1.38 Inhibin-b C SMAD1 NM_005900 1.55 SMAD family member 1 SMAD2 NM_005901 À1.87 1.28 À1.33 SMAD family member 2 SMAD3 NM_005902 11.57 1.60 SMAD family member 3 SMAD4 NM_005359 À1.58 1.41 SMAD family member 4 ID1 NM_181353 2.52 1.52 Inhibitor of DNA binding 1, dominant-negative helix–loop–helix protein ID3 NM_002167 1.25 1.66 Inhibitor of DNA binding 3, dominant-negative helix–loop–helix protein THBS1 NM_003246 11.62 À2.01 1.24 Thrombospondin 1 ZFYVE16 NM_014733 5.69 À1.39 Zinc finger, FYVE domain containing 16

Wnt signaling pathway WNT5A NM_003392 12.91 À1.28 Wingless-type MMTV integration site family, member 5A FZD5 NM_003468 2.96 3.03 Frizzled homolog 5 (Drosophila) FZD8 NM_031866 2.08 3.73 Frizzled homolog 8 (Drosophila) CTNNB1 NM_001904 4.90 1.56 Catenin (cadherin-associated protein)-b 1, 88 kDa JUN NM_002228 À2.02 1.52 Jun oncogene MAPK8 NM_139049 À1.37 1.51 Mitogen-activated protein kinase 8 (JNK1) MAPK9 NM_139070 1.63 Mitogen-activated protein kinase 9 (JNK2) NFAT5 NM_173215 2.50 À1.25 Nuclear factor of activated T-cells 5, tonicity-responsive

ErbB signaling pathway TGFA NM_003236 2.47 1.55 Transforming growth factor-a AREG NM_001657 À1.26 À1.96 1.50 Amphiregulin CDKN1B NM_004064 À2.57 1.26 Cyclin-dependent kinase inhibitor 1B (p27, Kip1) HBEGF NM_001945 1.90 À1.43 Heparin-binding EGF-like growth factor PAK2 NM_002577 À1.71 1.20 1.20 p21 protein (Cdc42/Rac)-activated kinase 2 SOS1 NM_005633 8.20 1.62 Son of sevenless homolog 1 (Drosophila) SOS2 NM_006939 5.87 À1.32 Son of sevenless homolog 2 (Drosophila)

Notch signaling pathway NOTCH1 NM_017617 1.53 Notch homolog 1, translocation-associated (Drosophila) NUMB NM_003744 1.92 Numb homolog (Drosophila) DTX3 L NM_138287 2.99 3.61 À1.34 Deltex 3-like (Drosophila) DVL2 NM_004422 À1.57 Dishevelled, dsh homolog 2 (Drosophila) JAG1 NM_000214 11.35 1.63 1.28 Jagged 1 (Alagille syndrome) KAT2A NM_021078 1.73 À1.31 K (lysine) acetyltransferase 2A

Jak-STAT signaling pathway JAK2 NM_004972 1.88 À2.06 À1.33 Janus kinase 2 (a protein tyrosine kinase) STAT1 NM_139266 10.88 2.16 À1.34 Signal transducer and activator of transcription 1, 91 kDa STAT2 NM_005419 4.51 À1.50 Signal transducer and activator of transcription 2, 113 kDa STAT3 NM_139276 4.24 À1.50 Signal transducer and activator of transcription 3 (acute phase) SOS1 NM_005633 8.20 1.62 Son of sevenless homolog 1 (Drosophila) SOS2 NM_006939 5.87 À1.32 Son of sevenless homolog 2 (Drosophila) SOCS4 NM_199421 À1.41 1.33 Suppressor of cytokine signaling 4 SOCS7 NM_014598 À1.76 1.34 Suppressor of cytokine signaling 7 GHR NM_000163 4.31 Growth IFNB1 NM_002176 À2.61 Interferon-b 1, fibroblast IFNGR1 NM_000416 8.70 1.68 Interferon-g receptor 1 IL15 NM_000585 3.40 À1.52 Interleukin 15 IL15RA NM_002189 2.06 À1.32 Interleukin 15 receptor-a LIFR NM_002310 1.68 1.54 Leukemia inhibitory factor receptor-a OSMR NM_003999 9.95 1.79 À1.34 Oncostatin M receptor

MAPK signaling pathway DUSP1 NM_004417 À1.25 Dual specificity phosphatase 1 DUSP10 NM_144729 2.25 2.65 À1.33 Dual specificity phosphatase 10 DUSP2 NM_004418 1.50 Dual specificity phosphatase 2 (EC:3.1.3.16 3.1.3.48) FGFR4 NM_022963 À3.81 1.52 Fibroblast growth factor receptor 4 (EC:2.7.10.1)

Oncogene MicroRNA-221/222 and fulvestrant-resistant breast cancer X Rao et al 1090 Table 1 Continued Gene symbol Genebank Fold-change Description

MCF7-F versus pre221/222 in si221/222 in MCF7 MCF7 MCF7-F

FLNA NM_001456 À1.29 À1.52 1.22 Filamin A-a (actin-binding protein 280) FLNB NM_001457 À2.70 Filamin B-b (actin-binding protein 278) GNA12 NM_007353 À3.47 À3.06 Guanine nucleotide-binding protein (G protein)-a 12 HSPA8 NM_153201 À1.89 1.25 Heat shock 70 kDa protein 8 IL1R1 NM_000877 À3.44 À2.31 Interleukin 1 receptor, type I MAP2K5 NM_145162 12.39 À1.31 Mitogen-activated protein kinase kinase 5 MAP3K4 NM_006724 À3.57 1.83 À1.21 Mitogen-activated protein kinase kinase kinase 4 MAP3K5 NM_005923 3.60 1.50 Mitogen-activated protein kinase kinase kinase 5 (EC:2.7.11.25) MAP3K8 NM_005204 8.35 1.54 Mitogen-activated protein kinase kinase kinase 8 (EC:2.7.11.25) MAPK8 NM_139049 À1.37 1.51 Mitogen-activated protein kinase 8 (JNK1) MAPK9 NM_139070 1.63 Mitogen-activated protein kinase 9 (EC:2.7.11.24) (JNK2) MAPT NM_173727 À1.69 À1.70 1.23 Microtubule-associated protein tau MKNK1 NM_198973 2.26 À1.86 À1.28 MAP kinase-interacting serine/threonine kinase 1 PLA2G10 NM_003561 2.02 1.84 1.42 Phospholipase A2, group X PLA2G12A NM_030821 À2.64 À1.54 Phospholipase A2, group XIIA (EC:3.1.1.4) PPM1A NM_177952 À2.98 1.22 Protein phosphatase 1A (formerly 2C), magnesium-dependent-a isoform PPP3CA NM_000944 À1.98 1.23 Protein phosphatase 3 (formerly 2B), catalytic subunit-a isoform (calcineurin A-a) PPP3R1 NM_000945 À1.82 1.37 Protein phosphatase 3 (formerly 2B), regulatory subunit B-a isoform PPP5C NM_006247 1.32 Protein phosphatase 5, catalytic subunit RASA2 NM_006506 3.95 À1.22 RAS p21 protein activator 2 STK4 NM_006282 5.37 À1.37 Serine/threonine kinase 4 TNFRSF1A NM_001065 4.67 À1.27 Tumor necrosis factor receptor superfamily, member 1A

p53 signaling pathway PERP NM_022121 2.35 À1.21 PERP, TP53 apoptosis effector PMAIP1 NM_021127 2.19 À1.58 Phorbol-12-myristate-13-acetate-induced protein 1 CASP3 NM_004346 À2.51 Caspase 3, apoptosis-related cysteine peptidase CDKN1A NM_078467 2.03 2.73 Cyclin-dependent kinase inhibitor 1A (p21, Cip1) FAS NM_000043 2.61 Fas (Tumor necrosis factor receptor superfamily, member 6) TNFRSF10B NM_147187 5.05 1.79 Tumor necrosis factor receptor superfamily, member 10b IGFBP3 NM_000598 265.29 2.03 Insulin-like growth factor-binding protein 3 GADD45A NM_001924 2.21 2.32 À1.42 Growth arrest and DNA-damage-inducible-a GADD45G NM_006705 À1.32 Growth arrest and DNA-damage-inducible-g SERPINE1 NM_000602 2.77 À3.04 Serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1 SESN1 NM_014454 3.03 1.51 Sestrin 1 SESN2 NM_031459 1.36 À1.34 Sestrin 2 THBS1 NM_003246 11.62 À2.01 1.24 Thrombospondin 1 SHISA5 NM_016479 3.23 1.69 À1.30 Shisa homolog 5 (Xenopus laevis) TP73 NM_005427 1.34 Tumor protein MDM2 NM_006882 13.22 À1.46 Mdm2 p53-binding protein homolog (mouse) PTEN NM_000314 À2.23 À1.54 Phosphatase and tensin homolog (EC:3.1.3.67 3.1.3.16 3.1.3.48)

Focal adhesion CAV1 NM_001753 À1.59 À20.66 1.30 Caveolin 1, caveolae protein, 22 kDa CAV2 NM_198212 À2.32 À7.38 1.38 Caveolin 2 CDC42 NM_044472 À2.24 1.46 Cell division cycle 42 (GTP-binding protein, 25 kDa) PAK1 NM_002576 À1.51 1.39 p21 protein (Cdc42/Rac)-activated kinase 1 PAK2 NM_002577 À1.71 1.29 p21 protein (Cdc42/Rac)-activated kinase 2 BCAR1 NM_014567 7.26 1.59 Breast cancer anti-estrogen resistance 1 BCL2 NM_000657 À1.88 B-cell CLL/lymphoma 2 DIAPH1 NM_005219 4.79 À1.25 Diaphanous homolog 1 (Drosophila) FLNA NM_001456 À1.29 À1.52 1.22 Filamin A-a (actin-binding protein 280) FLNB NM_001457 À2.70 Filamin B-b (actin-binding protein 278) PPP1CB NM_002709 À3.04 1.40 Protein phosphatase 1, catalytic subunit-b isoform (EC:3.1.3.16)

upregulated b-catenin-mediated gene transcription was used to inhibit b-catenin activity (Kim et al., (Figure 6c). To further investigate whether the increased 2006). Cell proliferation assays revealed that inhibiting b-catenin activity contributed to miR-221/222-mediated b-catenin activity blocked the growth of miR-221 estrogen-independent growth, epigallocatechin-3-gallate and/or miR-222-overexpressing MCF7 cells, but not

Oncogene MicroRNA-221/222 and fulvestrant-resistant breast cancer X Rao et al 1091

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 ¼ 2, with two independent experiments). *Po0.05, **Po0.01. parental MCF7 cells or scramble control-treated b-Catenin overexpression partially confers fulvestrant cells (Figure 6d). Taken together, these results implicate resistance miR-221/222 as a positive regulator of b-catenin Having demonstrated that miR221/222-activated expression and transcriptional activity, and the subse- b-catenin supports estrogen-independent growth, we quent transactivation of b-catenin target genes appears next examined whether b-catenin alone is enough to to contribute to the acquisition of estrogen-independent confer fulvestrant resistance. MCF7 cells were transi- growth. ently transfected with b-catenin-overexpressing plasmids

Oncogene MicroRNA-221/222 and fulvestrant-resistant breast cancer X Rao et al 1092

Figure 6 miR-221/222 upregulates and activates b-catenin. (a) Upregulated b-catenin mRNA in MCF7-T and BT474 cells. qRT–CR results indicated the time-course changes of b-catenin mRNA in MCF7-T cells (right panel) and the b-catenin mRNA level 48 h after transfection in BT474 cells (left panel) (mean±s.e., n ¼ 2, with two independent experiments). **Po0.01. (b) Increased b-catenin protein level in the nuclear fraction (MCF7, MCF7-T and BT474). Immunoblotting results showed the protein level of b-catenin in nucleus and cytoplasm, whereas TATA binding protein (TBP) was used as loading control for nuclear (upper panel) and b-tubulin as the control for cytoplasmic proteins (lower panel). (c) Increased b-catenin transcriptional activity. MCF7 cells were transfected with TOPflash or FOPflash reporter plasmids, together with b-gal pasmid for 48 h. Luciferase activities were measured using a luminometer, and b-gal assays were performed to normalize transfection efficiencies. (mean±s.e., n ¼ 3). *Po0.05. (d) Inhibition of b-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 ¼ 6). *Po0.05, **Po0.01.

and then treated with varying doses of fulvestrant. Cell vector-treated cells or parental cells (Figure 7a). Immuno- proliferation assays indicated that although b-catenin blotting analysis indicated that overexpression of b-catenin alone was not able to increase cell growth in estrogen- occurred mostly in the cytoplasmic fraction (Figure 7b), free medium (Supplementary Figure S11), cells over- suggesting the existence of other factors in MCF7 that expressing b-catenin were more vulnerable to fulves- repress b-catenin nuclear translocation. These results trant-induced growth inhibition compared with empty demonstrated that although b-catenin contributes to

Oncogene MicroRNA-221/222 and fulvestrant-resistant breast cancer X Rao et al 1093

Figure 7 Overexpression of b-catenin partially confers MCF7 cells resistance to fulvestrant. (a) Decreased fulvestrant sensitivity of b-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 ¼ 6). **Po0.01. (b) Immunoblotting results indicating the protein level of b-catenin.

Figure 8 miR-221/222 alter cell sensitivity to TGF-b1-induced growth inhibition. (a) TGF-b1 sensitivity of MCF7 cells. Cells were treated with doses of TGF-b1 for 4 days and cell growth rates (drug versus vehicle) were determined by MTT assay. (mean±s.e., n ¼ 6). *Po0.05, **Po0.01. (b) TGF-b1 sensitivity of MCF7-F cells determined as in (a). acquired fulvestrant resistance, b-catenin overexpression cancer (Zugmaier et al., 1989; McEarchern et al., 2001), alone was not sufficient for estrogen-independent miR-221/222 may serve as an important mediator of the growth, further suggesting a requirement for additional transition of TGF-b from growth suppressor to tumor miR-221/222-regulated genes. promoter. miR-221/222 modulate the effects of TGF-b An association between TGF-b signaling pathway and Discussion antiestrogen resistance has been reported (Buck and Knabbe, 2006). Based on our expression profiling results Acquired fulvestrant resistance is associated with loss of showing that miR-221/222-regulated genes in MCF7 ERa expression, E2-independent growth, and activation and MCF7-F cells were enriched for the TGF-b path- of multiple growth-stimulatory pathways (Nicholson way (Figure 4) and cell proliferation assays revealing et al., 2005; Normanno et al., 2005; Fan et al., 2006). a growth inhibitory effect of TGF-b1 on MCF7 but not Recent studies (Miller et al., 2008; Zhao et al., 2008), MCF7-F cells (Supplementary Figure S12) (Kalkhoven including our own (Xin et al., 2009), suggest a et al., 1995; Sovak et al., 1999), we hypothesized that prominent role of miR-221 and -222 in the acquisition the altered sensitivity to TGF-b-induced growth inhi- of antiestrogen resistance. In the current study, we bition was associated with miR-221/222 expression. demonstrate that marginal miR-221/222 upregulation To examine this possibility, pre-miRNA-treated MCF7 (B2-fold) is associated with tamoxifen resistance but cells were treated with TGF-b1. Overexpression of miR- marked overexpression (up to 25-fold) of miR-221/222 221 and/or miR-222 increased MCF7 cell survival is characteristic of fulvestrant resistance (Figure 1a). (versus scramble control; Figure 8a), and knockdown Ectopic expression of miR-221/222 counteracts the well- of miR-221 and/or miR-222 in MCF7-F cells resulted in known effects of E2 starvation and fulvestrant-induced growth inhibition by TGF-b1 (Figure 8b). As TGF-b cell damage and death in fulvestrant-sensitive cells, can act as a tumor suppressor or enhancer in breast whereas strikingly increasing cellular proliferation and

Oncogene MicroRNA-221/222 and fulvestrant-resistant breast cancer X Rao et al 1094 conferring SERD resistance (Figure 2). However, down- FZD5 expression (Table 1), may activate Wnt signaling regulation of miR-221/222 significantly suppresses cell and/or facilitate b-catenin nuclear accumulation by growth and clonogenic activity of fulvestrant-resistant repressing Wnt pathway inhibitors AXIN2, SFRP2, cells (Figure 3). Interestingly, both E2 and tamoxifen CHD8 and NLK, the predicted targets of miR-221/222 repress expression of endogenous miR-221/222 in (determined using DIANA microT 4.0 (Maragkakis MCF7 cells, but fulvestrant dramatically induces miR- et al., 2009)). The observation that miR-221/222-activated 221/222 (Figure 1b), suggesting that constitutive upre- b-catenin supports estrogen-independent growth (Figures 6c gulation of miR-221/222 seen in fulvestrant-resistant and d), whereas overexpressing b-catenin alone does not cells is likely initiated by prolonged SERD treatment, (or even decreases cell proliferation under estrogen-free attenuating the ERa/E2-mediated repression of miR- conditions; Supplementary Figure S11), further indicates 221/222, in accord with our recent study on a negative that miR-221/222 trigger multiple events, which are miR-221/222-ERa regulatory loop (Di Leva et al., required for the development of SERD resistance: 2010). As it is well established that maintenance of a b-catenin contributes to decreased fulvestrant sensiti- strong ERa-positive phenotype is characteristic of vity, whereas other factors further promote autocrine- tamoxifen-resistant breast cancer cells (Wijayaratne regulated proliferation. et al., 1999; Fan et al., 2006), upregulation of miR-221/ The most commonly regulated pathway by miR-221/ 222 in tamoxifen-resistant cells (Figure 1a; Miller et al., 222 in MCF7 and MCF7-F cells is the TGF-b signaling 2008) must be due to a different mechanism(s). Interest- pathway (Figure 4), with increased TGFBR2, BMPR2 ingly, upregulation of these two miRNAs has recently and SMAD2 after transfection (24–48 h; Figures 5b and been reported in CD44 þ CD24À/lowlineageÀ human breast c). SMADs (SMAD1/2/3), key regulators of TGF-b cancer stem cells (Shimono et al., 2009), which are signaling activity, are upregulated by miR-221/222; believed to be largely responsible for propagating the however, miR-221/222 also induced two TGF-b-re- drug-resistant phenotype (O’Brien et al., 2009), further pressed genes, ID1 and ID3 (Table 1), in accord with stressing the importance of miR-221/222 in breast cancer downregulated TGF-b signaling activity in MCF7-F progression and acquisition of antiestrogen resistance. cells (with high miR-221/222 expression, Supplementary By analyzing miR-221/222-regulated gene expression Figure S8 and Figure 1a). Owing to the great diversity of profiles (knockdown of miR-221/222 in FR and over- cell-specific gene responses elicited by TGF-b family expression of miR-221/222 in MCF7), we demonstrate members (Massague, 2000), whether the TGF-b path- that multiple oncogenic signaling pathways are regu- way is activated or depressed by miR-221/222 is not lated by miR-221/222 (Figure 4), which we (Fan et al., clear, but the growth inhibitory effect of TGF-b is 2006) and others (Nicholson et al., 2005; Normanno suppressed by miR-221/222 (Figure 8). As a potent et al., 2005) have reported contribute to the acquisition inhibitor of primary human mammary epithelial cells of fulvestrant resistance. One of the most strikingly and most breast cancer cell lines (Zugmaier et al., 1989; regulated is the Wnt pathway, showing upregulation of Basolo et al., 1994), TGF-b is activated by antiestrogens CTNNB1 (b-catenin) and FZD5 12 h after miR-221/222 and participates in antiestrogen-induced growth inhibi- overexpression (Figures 5a and 6a). As miRNAs can tion (Buck et al., 2004). However, by stimulating also upregulate target gene expression (Vasudevan angiogenesis, inducing extracellular matrix degradation, et al., 2007; Chen et al., 2010), these may represent increasing invasion and metastasis, and inhibiting direct miR-221/222targets. However, using two target antitumor immune responses, TGF-b can promote prediction programs (TargetScan (Lewis et al., 2003) tumor progression during latter stages (Gorsch et al., and PITA (Kertesz et al., 2007)), we were not able to 1992; McEarchern et al., 2001). Also, defects in identify seed sequence binding sites for miR-221/222 in autocrine growth-inhibitory loops involving TGF-b b-catenin and FZD. In addition, mRNA stability assays signal transduction have been recognized as playing a using 5,6-dichloro-1–D-ribofuranosylbenzimidazole de- central role in the development of antiestrogen resis- monstrated that b-catenin upregulation occurs at the tance (Buck and Knabbe, 2006). Thus, miR-221/222 transcriptional level (data not shown). Thus, the may serve as a switch to turn off the tumor-suppressing mechanism of miR-221/222-induced activation of effects of TGF-b and enhance its tumor-promoting b-catenin transcription remains unclear. Interestingly, function during the acquisition of fulvestrant resistance. miR-221/222 also dramatically increases b-catenin p53 signaling is also among the pathways that are protein levels, particularly in the nuclear fraction activated by miR-221/222 (Figure 4), with upregulation (Figure 6b), which is not achievable by introducing of several p53 target genes, including p21Cip1, PERP, b-catenin-overexpressing plasmids alone (Figure 7b). CASP3, FAS, IGFBP3, GADD45A and SESNs (SES- b-Catenin is known to be sequestered in the cyto- TRINs) (Table 1). MCF7-F cells having elevated miR- plasm by the destruction complex APC, AXIN, GSK-3b 221/222 also show higher p53 signaling activity com- and CK1a, resulting in proteasomal degradation. pared with MCF7 (Supplementary Figure S8, Engagement of Wnt receptors inhibits destruction Figure 1a). Oncogene activation induced by miR-221/ complex formation, allows for b-catenin release, trans- 222 (Table 1) and altered cell morphology (reduced cell– location to the nucleus and subsequent activation of cell contact, Figure 2c) might drive the activation of p53 b-catenin target gene transcription (Takahashi-Yanaga (Vousden and Prives, 2009). Although the first identified and Kahn, 2010). In addition to upregulating b-catenin tumor suppressor, additional roles for p53 have recently transcription, miR-221/222, by increasing Wnt5A and been reported, including regulation of metabolism,

Oncogene MicroRNA-221/222 and fulvestrant-resistant breast cancer X Rao et al 1095 acting as antioxidants and even the antiapoptotic miR-221 and miR-222 (20-O-Me-221-GAAACCCAGCAGA potential to increase cell survival (Janicke et al., 2008; CAAUGUAGCUL and 20-O-Me-222-GAGACCCAGUAGC Vousden and Prives, 2009). miR-221/222 may utilize CAGAUGUAGCUL), as well as a negative control antagomir these newly ascribed-p53 functions to confer fulvestrant (20-O-Me-enhanced green fluorescent protein (eGFP)-AAGG resistance. For example, p21Cip1 may also function to CAAGCUGACCCUGAAGUL) (Visone et al., 2007), were inhibit apoptosis, and GADD45A and SESTRINs allow synthesized by Eurofins MWG Operon (Huntsville, AL, USA). Other biologicals used in this study have been described cells to survive until cellular damage has been resolved previously (Fan et al., 2006). BT474 cells were purchased from (Vousden and Prives, 2009). Interestingly, PTEN, a American Type Culture Collection (Manassas, VA, USA). See recently identified miR-221/222 target (Garofalo et al., Supplementary materials for antibodies and plasmids. 2009), was not regulated by miR-221/222 in MCF7 cells but slightly induced by miR-221/222 knockdown in Cell culture, transfection and clonogenicity assays MCF7-F cells (Supplementary Figure S13), implying MCF7 and MCF7-F cells were cultured as previously cell-specific regulation by miR-221/222. described (Fan et al., 2006). BT474 cells were cultured In the focal adhesion pathway, two major compo- according to American Type Culture Collection- suggested nents of the caveolae plasma membranes, CAV1 and conditions. Pre-miRNAs or antagomirs were transfected with CAV2 (Table1 and Supplementary Figure S9), were Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) accord- downregulated by miR-221/222, consistent with our ing to the manufacturer’s instructions. Plasmids were trans- previous report (Di Leva et al., 2010). Although a fected with Fugene HD Transfection Reagent (Roche predicted target of miR-221/222 (TargetScan (Lewis Diagnostics Co., Indianapolis, IN, USA) according to the et al., 2003)), downregulation of CAV2 was not observed manufacturer’s instructions. The clonogenic activity was examined as previously described (Fan et al., 2006). See until 72 h, but miR-221/222 overexpression rapidly (12 h) Supplementary materials for details and other methods used. downregulated CAV1, despite the fact that CAV1 lacks a perfect seed sequence miR-221/222 binding site. The mechanism(s) underlying this phenomenon requires further Microarray data analysis To examine global gene expression profiles, MCF7-F cells investigation. As expression of both CAV1 and CAV2 has were treated with miR-221 or miR-222 antagomirs or scramble been shown to inversely correlate with HER2/neu expres- control for 72 h, followed by RNA preparation/labeling and sion and tumor size in human breast cancer (Sagara et al., hybridization to Affymetrix GeneChip HG-U133 plus 2.0 2004), CAV1 and CAV2 downregulation may increase cell arrays, according to Affymetrix standard protocols (Affyme- proliferation and enhance epidermal growth factor receptor trix, Santa Clara, CA, USA). Gene expression data were then (EGFR) signaling activity. processed and analyzed using Affymetrix Microarray Analysis miR-221/222 have been considered to be oncogenes or Suite version 5.0 (MAS5), as described previously (Fan et al., tumor suppressors, depending on tumor type (Croce, 2006; Li et al., 2009), and deposited in Gene Expression 2009). Based on our results, ERa status may serve as a Omnibus (accession number: GSE19777). See Supplementary marker for the role of miR-221/222 in breast cancer. In materials for details of further analysis. ERa-positive cells, which depend on E2/ERa signaling for proliferation, fulvestrant treatment (Figure 1b) or E2 Statistical analysis starvation (Di Leva et al., 2010) initially induce miR- For MTT cell viability, BrdU cell proliferation, clonogenicity 221/222, which function as tumor suppressors by and qRT–PCR assays, statistical significance was analyzed by unpaired Student’s t test. P-values o0.05 were considered downregulating the receptor (Figure 2a). However, statistically significant. prolonged inhibition of ERa activity by fulvestrant treatment or E2 starvation releases miR-221/222 from the negative regulatory loop, resulting in constitutive upregulation of miR-221/222. In this scenario, miR-221/ Conflict of interest 222 act as ‘oncomirs’ by targeting cell cycle inhibitors (p27Kip1,p57Kip2), other tumor suppressors (Garofalo The authors declare no conflict of interest. et al., 2009), activating oncogenic signaling pathways (including Wnt, Notch and MAPK), and modulating Acknowledgements TGF-b and p53 signaling to support ERa-independent proliferation and promote tumor progression. Targeting We thank Dr Man-Wook Hur for providing the TOPflash and these two oncomirs may be a potential therapeutic FOPflash plasmids, Jim Powers for assistance with fluorescence strategy for preventing the development of fulvestrant microscopy, Dr Meiyun Fan for advice on experiments and resistance or resensitizing breast tumors to this potent Dr Curt Balch for critical comments and paper preparation. and effective estrogen antagonist. The IUB Light Microscopy Imaging Center provided micro- scopy resources. The microarray studies were carried out using the facilities of the Center for Medical Genomics (Dr H Edenberg, Materials and methods Director and Dr J McClintick) at Indiana University School of Medicine. The Center for Medical Genomics is supported in part Materials and cell lines by the Indiana Genomics Initiative of Indiana University, which is Pre-miR miRNA precursor for ectopic expression of miR-221, supported in part by the Lilly Endowment, Inc. This work was miR-222 and the scramble control were purchased from supported in part by NIH grants CA085289, CA113001 and Applied Sciences/Ambion (Austin, TX, USA). Chemically CA125806, American Cancer Society Research Scholar Grant stabilized antisense miRNA inhibitors (antagomirs) targeting 09-244-01, and the Walther Cancer Foundation (Indianapolis).

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