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

Abstract

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) α-negative (ERα−) vs. ERα-positive (ERα+) breast cancer cell lines, and in ERα− breast tumors. In MCF-7 cells the low level of Ago2 was found to be dependent upon active ERα/estrogen signaling. Interestingly, the high expression of Ago2 in ERα− 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 ERα+ 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. Argonaute-2 is elevated in ERα- breast cancer cells due to epidermal growth factor receptor/MAPK signaling, and overexpression of this gene induces a more transformed phenotype in ERα+ MCF-7 cells.