P3-03-05: Identification of miRNAs and Their Associated Target Genes Involved in Endocrine Resistance in Breast Cancer.
Authors: Healy NA , Creighton CJ , Fu X , Tsimelzon A , Hilsenbeck SG , Miller N , Kerin MJ , Osborne CK , Schiff R 
Published: June 2012

Abstract
Background: Despite the widespread use of the selective estrogen receptor modulator tamoxifen or strategies of estrogen deprivation in the treatment of estrogen receptor-positive breast cancer, tumor resistance to endocrine therapy remains a serious clinical problem. MiRNAs are a class of endogenous, single stranded RNA molecules that play a pivotal role in the regulation of gene expression. Altered expression levels of these molecules have been implicated in a number of malignancies, including breast cancer. The aim of this study was to identify those miRNAs that may be associated with endocrine resistance and to further investigate their role in development of this resistance.
Methods: MCF7L breast cancer cells were cultured in regular parental medium (Par) or in phenol-red free, 10% charcoal-stripped FBS medium treated by estrogen deprivation (ED, &gt;6months), tamoxifen (Tam, 10−7M, &gt;6months), or estrogen (E2, 10−9M,&gt;1month). Cells treated by long-term Tam or ED developed resistance (TamR or EDR) and resumed cell growth, which was confirmed by growth curve assay. Parallel short-term treatment (10 days) with estrogen deprivation or tamoxifen to represent the endocrine-sensitive phase, and short-term E2 treated cells as a control was also performed. A whole-transcriptome miRNA microarray was performed by High Throughput Genomics (Tucson, AZ), using cell lysates from Par, short-term/endocrine sensitive, E2 treated cells, TamR and EDR. The expression levels of several miRNAs were validated by real-time quantitative PCR (Q-PCR). MiRNAs dysregulated in TamR cells were integrated with predicted gene targets showing anti-correlated expression patterns across human breast tumors in The Cancer Genome Atlas (TCGA) datasets.
Results: The miRNA microarray identified 23 miRNAs that are upregulated in TamR cells (&gt;1.7 fold change, p&lt;0.005) compared to parental cells grown in the presence of E2, and 37 miRNAs that were downregulated (&gt;50%, p&lt;0.005). In addition, 6 miRNAs were observed to be upregulated in EDR and 3 downregulated in EDR compared with controls. Validation of four of these upregulated miRNAs within the TamR group, miR221, miR-222, miR-301b and miR-181c, was performed using Q-PCR. Previously identified genes associated with TamR were scanned for potential binding sites of these deregulated miRNAs using miRanda and TargetScan and many potential miRNA targeted genes are downregulated in TamR. Both miR-221 and miR-181c are predicted to target estrogen receptor gene (ESR1), and downregulation of this target gene was confirmed at the mRNA level by Q-PCR. A strong negative correlation was identified between ESR1 and both miR-181c and miR-221 expression levels in breast tumor samples, according to TCGA data. Work is ongoing to identify the role of miR-181c and other miRNAs in tamoxifen resistance, using a loss-of-function approach. Parallel miRNA profiling of multiple endocrine models is underway to identify commonly deregulated miRNAs associated with endocrine resistance.
Conclusions: This study has identified those miRNAs associated with endocrine resistance in breast cancer. These miRNAs may provide potential predictive markers of resistance to endocrine therapy and modulation of these may lead to increased therapeutic sensitivity.
Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-03-05.