A Regulatory “miRcircuitry” Involving miR221&222 and ERα Determines ERα Status of Breast Cancer Cells

Estrogen Receptor α (ERα) is the most important diagnostic and prognostic factor in breast cancer. Several lines of evidence have suggested that ERα-negative breast tumors, highly aggressive and non-responsive to hormonal therapy, arise from ERα-positive precursors through different molecular pathways. microRNAs (miRs) are small non-coding RNAs that regulate gene expression at post-transcriptional level and are aberrantly expressed in breast cancer. We hypothesized that microRNAs may have a pivotal role in ERα suppression and ERα-negative tumors formation. MicroRNAs microarray, quantitative real-time PCR, immunohistochemistry and in-situ hybridization analyses of breast cancer cell lines and primary tumors with different ERα-status indicated that miR221&222 are exclusively expressed in ERα-negative breast tumors. Overexpression of both microRNAs in ERα- positive cell lines strongly reduces the levels of ERα protein and, using luciferase reporter assays, we clearly demonstrated that ERα is a bona fide target of miR221&222. Gene expression profiles were analyzed after miR221 or miR222 over-expression in ERα-positive cells: up-regulation of anti-apoptotic genes, growth factors and down modulation of adhesion molecules indicated that miR221&222 may not only increase the ability to suppress apoptosis and accelerate tumor formation but also promote remodeling of the tumor microenvironment and escape of tumor cells through the basement membrane, all characteristics of ERα-negative cells. To gain more insights into the inverse correlation between miR221&222 and ERα in breast cancer, by computational analyses and reporter assay, we characterized the transcriptional unit of miR221&222. We identified several estrogen-responsive elements (ERE) at the genomic locus of miR221&222 and, by using different approaches, we demonstrated that ERα suppresses miR221&222 expression. Overexpression of exogenous or endogenous ERα protein in ERα-negative cells markedly repressed miR221&222; conversely, knockdown of ERα in ERα-positive cells increased their expression. Finally, chromatin immunoprecipitation (ChIP) on ERα-positive cells showed that ligand-bound ERα and its co-repressor proteins, NcoR and SMRT, were highly enriched at miR221&222 genomic locus. Notably, ChIP experiments after estradiol (E2) stimulation or ERα-knockdown revealed that ERα recruitment is essential for the corepressor enrichment and miR221&222 repression. These findings suggest that the negative regulatory loop involving miR221&222 and ERα may confer proliferative advantage and migratory activity to breast cancer cells and promote the transition from ERα-positive to ERα-negative tumors. Therefore, the elucidation of this pathway could be an important step in the development of the next generation of breast cancer therapeutics agents that may prevent hormone-resistance that frequently occurs during treatment.