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Argonaute-2 Expression Is Regulated by Epidermal Growth Factor Receptor and Mitogen-Activated Protein Kinase Signaling and Correlates with a Transformed Phenotype in Breast Cancer Cells

Department of Cell Biology (B.D.A., B.A.W.) and Center for Vascular Biology (K.P.C.), University of Connecticut Health Center, Farmington, Connecticut 06030

Address all correspondence and requests for reprints to: Bruce White, Department of Cell Biology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, Connecticut 06030-3505. E-mail: BWhite{at}nso2.uchc.edu.

Argonaute (Ago) 2 is the catalytic engine of mammalian RNA interference, but little is known concerning the regulation of Ago2 by cell-signaling pathways. In this study we show that expression of Ago2, but not Ago1, Ago3, or Ago4, is elevated in estrogen receptor (ER) -negative (ER^–) vs. ER-positive (ER⁺) breast cancer cell lines, and in ER^– breast tumors. In MCF-7 cells the low level of Ago2 was found to be dependent upon active ER/estrogen signaling. Interestingly, the high expression of Ago2 in ER^– cells was severely blunted by inhibition of the epidermal growth factor (EGF) receptor/MAPK signaling pathway, using either a pharmacological MAPK kinase inhibitor, U0126, or a small interfering RNA directed against EGF receptor. Half-life studies using cycloheximide indicated that EGF enhanced, whereas U0126 decreased, Ago2 protein stability. Furthermore, a proteosome inhibitor, MG132, blocked Ago2 protein turnover. The functional consequences of elevated Ago2 levels were examined by stable transfection of ER⁺ MCF-7 cells with full-length and truncated forms of Ago2. The full-length Ago2 transfectants displayed enhanced proliferation, reduced cell-cell adhesion, and increased migratory ability, as shown by proliferation, homotypic aggregation, and wound healing assays, respectively. Overexpression of full-length Ago2, but not truncated forms of Ago2 or an empty vector control, reduced the levels of E-cadherin, β-catenin, and β-actin, as well as enhanced endogenous miR-206 activity. These data indicate that Ago2 is regulated at both the transcriptional and posttranslational level, and also implicate Ago2 and enhanced micro-RNA activity in the tumorigenic progression of breast cancer cell lines.