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Published OnlineFirst November 11, 2014; DOI: 10.1158/0008-5472.CAN-14-1041 Cancer Tumor and Stem Cell Biology Research Loss of Estrogen-Regulated microRNA Expression Increases HER2 Signaling and Is Prognostic of Poor Outcome in Luminal Breast Cancer Shannon T. Bailey1,2,3, Thomas Westerling1,2,3, and Myles Brown1,2,3 Abstract Among the genes regulated by estrogen receptor (ER) are Genome Atlas (TCGA). Among luminal tumors, these miRNAs miRNAs that play a role in breast cancer signaling pathways. To are expressed at higher levels in luminal A versus B tumors. determine whether miRNAs are involved in ER-positive breast Although their expression is uniformly low in luminal B tumors, cancer progression to hormone independence, we profiled the they are lost only in a subset of luminal A patients. Interestingly, expression of 800 miRNAs in the estrogen-dependent human this subset with low expression of these miRNAs had worse breast cancer cell line MCF7 and its estrogen-independent deriv- overall survival compared with luminal A patients with high ative MCF7:2A (MCF7:2A) using NanoString. We found 78 expression. We confirmed that miR125b directly targets HER2 miRNAs differentially expressed between the two cell lines, and that let-7c also regulates HER2 protein expression. In addi- including a cluster comprising let-7c, miR99a, and miR125b, tion, HER2 protein expression and activity are negatively corre- which is encoded in an intron of the long noncoding RNA lated with let-7c expression in TCGA. In summary, we identified LINC00478. These miRNAs are ER targets in MCF7 cells, and an ER-regulated miRNA cluster that regulates HER2, is lost with nearby ER binding and their expression are significantly progression to estrogen independence, and may serve as a bio- þ decreased in MCF7:2A cells. The expression of these miRNAs marker of poor outcome in ER luminal A breast cancer patients. was interrogated in patient samples profiled in The Cancer Cancer Res; 75(2); 436–45. Ó2014 AACR. Introduction been demonstrated to play important roles in normal develop- ment and physiology as well as regulating a number of disease The estrogen receptor (ER) is an estrogen-regulated transcrip- processes including breast cancer (7–9). miRNAs have been tion factor that controls the transcription of numerous coding and þ reported to be generally downregulated in cancers, and their loss noncoding RNAs and is a key target for therapy in ER breast leads to the increased expression of targeted genes, notably cancers (1, 2). In breast cancer, ER acts predominantly by binding including oncogenes that lead to cancer progression. In breast to distal enhancer sites to mediate transcription (3). Downstream cancer, a number of miRNAs have been reported to be abnormally effectors of ER activity in breast cancer include genes with pro- regulated (10–13). ER has also been reported to regulate the oncogenic functions including survival and growth. It has been expression of a number of miRNAs in response to its ligand known for more than 40 years that a primary determinant of the estradiol (E2; refs. 14–17). response of breast cancers to endocrine therapy is the expression þ Here, we report the identification of miRNAs directly regulated of ER, leading to the first stratification of breast cancer into ER þ À by ER and differentially expressed in the estrogen-dependent ER and ER subsets. More recently, refined subsets have been iden- breast cancer cell line MCF7 and its hormone-independent deriv- tified by gene expression profiles characteristic of clinical subtypes ative MCF7:2A. The let-7c/miR99a/miR125b cluster is expressed in which ER may play different roles (4–6). in MCF7 cells where it is directly targeted by ER and both miRNAs are small noncoding RNAs approximately 22 bp in expression and ER binding are lost in MC7:2A cells. Expression length that regulate the expression of genes by targeting the 30 of this miRNA cluster is uniformly low in luminal B breast cancers, untranslated regions (UTR) of mRNAs. These molecules have which have a worse outcome than luminal A. Within the luminal A subtype, low expression of the cluster predicts for poor patient 1Center for Functional Cancer Epigenetics, Dana-Farber Cancer Insti- outcome. We find that two members of the cluster, let-7c and tute, Boston, Massachusetts. 2Department of Medical Oncology, mirR-125b, inhibit HER2 protein expression and increased 3 Dana-Farber Cancer Institute, Boston, Massachusetts. Department expression of the HER2 protein in luminal A tumors lacking of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts. expression of these miRNA may mediate their poor outcome. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Materials and Methods Corresponding Author: Myles Brown, Harvard Medical School, Center for Cell culture and reagents Functional Cancer Epigenetics, Dana-Farber Cancer Institute, 450 Brookline MCF7 cells were grown in high-glucose DMEM (Invitrogen) Avenue, D730, Boston, MA 02215. Phone: 617-632-3948; Fax: 617-582-8501; supplemented with 2 mmol/L L-glutamine, 10% (vol/vol) heat- E-mail: [email protected] inactivated FBS, 100 IU/mL penicillin, and 100 mg/mL strepto- doi: 10.1158/0008-5472.CAN-14-1041 mycin (Invitrogen) in a humidified incubator at 37C and 5% Ó 2014 American Association for Cancer Research. CO2. The MCF7:2A, MCF7:5C, and MCF7:LTLT cell lines were 436 Cancer Res; 75(2) January 15, 2015 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst November 11, 2014; DOI: 10.1158/0008-5472.CAN-14-1041 ER-Regulated miRNA Expression and Breast Cancer Outcome grown in phenol red-free high-glucose DMEM (Invitrogen) gene as a reporter with Lipofectamine 2000 following the man- supplemented with 2 mmol/L L-glutamine, 5% (vol/vol) heat- ufacturer's instructions. After incubation for 48 hours, the cells inactivated FBS, 100 IU/mL penicillin, and 100 mg/mL strepto- were lysed in 1Â Passive Lysis Buffer and assayed with the Dual- mycin (Invitrogen). The MCF7:LTLT cells were also supplemented Luciferase Reporter Assay System (Promega) to measure the with 1 mmol/L letrazole. The MCF7:2A and MCF7:5C cell lines Renilla luciferase activity and that of firefly luciferase, which served were obtained from V. Craig Jordan (Department of Oncology, as a transfection control. Lombardi Cancer Center, Georgetown University Medical Center, Washington, DC) and the MCF7:LTLT cell line was obtained Ago1 RNA immunoprecipitation from Angela Brodie (Department of Pharmacology and Experi- The Ago1 complex was immunoprecipitated as described in mental Therapeutics, University of Maryland School of Medicine, ref. 19. Briefly, A total of 2 Â 106 MCF7 and MCF7:2A cells in the Baltimore, MD). The Dharmacon anti-miRs and miRNA mimics growth phase were seeded in 10 cm plates. After 24 hours, the cells were obtained from ThermoFisher. were harvested in 400 mL lysis buffer (100 mmol/L KCl, 5 mmol/L MgCl2, 10 mmol/L HEPES, pH 7.0, 0.5% Nonidet P-40) supple- NanoString mented with 100 U/mL RNase Out (Invitrogen Cat# 10777-019) A total of 2 Â 106 MCF7 and MCF7:2A cells growing in the and Complete Protease Inhibitor Cocktail (Roche). The lysates exponential phase were seeded in 6-well plates and cultured for 2 were centrifuged, and 50 mL was set aside for input. days. The cells were then harvested for total RNA using the A total of 2 mg anti-Ago1 antibody (Abcam #ab5070) was miRNeasy Kit (Qiagen). A total of 100 ng of total RNA was prebound to protein A Dynabeads (Life Technologies). The anti- assayed using the Human nCounter miRNA Assay 2.0 Kit follow- body and lysate mixture was incubated overnight at 4C. The next ing the manufacturer's instructions (NanoString). Differences in morning, the beads were collected by magnetic separation, and miRNA expression were analyzed using the NanoSTRIDE software they were treated with DNaseI in NT2 buffer (50 mmol/L Tris, program (18) with default settings. Clustering of the differentially pH 7.4, 150 mmol/L NaCl, 1 mmol/L MgCl2, and 0.05% Nonidet expressed genes and heatmap generation was performed using the P-40) for 10 minutes at 37C. The beads were then washed twice GenePattern Server (genepattern.broadinstitute.org). The volcano with NT2 buffer, treated with proteinase K to digest protein, and plot displaying the significance of the miRNA differences was resuspended in 300 mL acid-phenol:chloroform (Ambion). The produced using R version 3.0.2. solution was centrifuged for 1 minute at 14,000 rpm at room temperature, the upper layer was collected, and the RNA was RT-PCR ethanol precipitated in the presence of GlycoBlue (Life Technol- For RT-PCR, total RNA was isolated using a combination of ogies, Inc.). The obtained RNA was resuspended in 30 mL water TRIzol (Sigma) and the RNeasy Mini Kit (Qiagen). First-strand and used to generate cDNA and subsequent RT-PCR analysis. cDNA, which was created using the Quantitect Reverse Transcription Kit (Qiagen) following the manufacturer's protocol, was assayed Transfection and immunoblotting using TaqMan miRNA assays (Life Technologies, Inc.), and the level MCF7 and MCF7:2A cells were transfected with 20 pmol of of U6 RNA was used as a control. The expression of LINC00478 was miRIDIAN miRNA anti-miRs or miRNA mimics as described measured using the Power SYBR Green PCR Master Mix (Life above. Cells were incubated for 5 days, and whole-cell extracts 0 Technologies, Inc.) with the following primers: 5 -GATCTGAGAA- were then harvested in RIPA buffer (TBS, 1% Nonidet P-40, 0.5% 0 0 CGCTGTCTGG-3 (forward) and 5 -AGAGTCTCCCTCCTGCTTCC- sodium deoxycholate, 0.1% SDS, 0.004% sodium azide).