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

• Published in: Endocrinology (Philadelphia) 2009-01, Vol.150 (1), p.14-23
• Main Authors: Adams, Brian D, Claffey, Kevin P, White, Bruce A
• Format: Article
• Language: English
• Subjects: Actin; Argonaute 2 protein; Argonaute Proteins; Biological and medical sciences; Breast cancer; Breast Neoplasms - genetics; Breast Neoplasms - pathology; Cell adhesion; Cell Aggregation; Cell Movement; Cell proliferation; Cell Transformation, Neoplastic - genetics; Cycloheximide; Cycloheximide - pharmacology; Disease Progression; E-cadherin; Epidermal growth factor; ErbB Receptors - physiology; Estrogen receptors; Estrogens; Eukaryotic Initiation Factor-2 - genetics; Extracellular Signal-Regulated MAP Kinases - metabolism; Female; Fundamental and applied biological sciences. Psychology; Gene expression; Gene Expression Regulation, Neoplastic; Growth factors; Gynecology. Andrology. Obstetrics; Humans; Kinases; Low level; Mammary gland diseases; MAP kinase; Medical sciences; MicroRNAs - genetics; Phenotype; Phenotypes; Polymerase Chain Reaction; Protein turnover; Proteins; Receptors; Ribonucleic acid; RNA; RNA, Messenger - genetics; RNA, Small Interfering - genetics; RNA-mediated interference; Signal transduction; siRNA; Transfection; Tumor cell lines; Tumors; Vertebrates: endocrinology; Wound Healing; β-Catenin
• ISSN: 0013-7227; 1945-7170
• DOI: 10.1210/en.2008-0984

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.