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) alpha-negative (ERalpha(-)) vs. ERalpha-positive (ERalpha+) breast cancer cell lines, and in ERalpha(-) breast tumors. In MCF-7 cells the low level of Ago2 was found to be dependent upon active ERalpha/estrogen signaling. Interestingly, the high expression of Ago2 in ERalpha(-) 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 ERalpha+ 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, beta-catenin, and beta-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.

miRNA biogenesis: Biological impact in the development of cancer

“…Interestingly, the altered expression of Ago2 is also associated with a transformed phenotype in breast cancer cells. 129 Hypoxia contributes to altered gene expression in tumors. In addition to affecting the activity of coding genes, increased levels of hypoxia in cancer cells also affects miRNA maturation and stability.…”

T cell activation induces proteasomal degradation of Argonaute and rapid remodeling of the microRNA repertoire

“…In addition, the stability and activity of the Drosha-DGCR8, Dicer-TRBP, and miRNA-induced silencing (miRISC) complexes are subject to regulation (Ghodgaonkar et al, 2009;Han et al, 2009;Paroo et al, 2009). Ago proteins in particular can undergo a variety of posttranslational modifications that affect their stability (Adams et al, 2009;Qi et al, 2008;Rybak et al, 2009;Rüdel et al, 2011). This is important because they can be a limiting factor for global miRNA expression levels (Diederichs and Haber, 2007;O'Carroll et al, 2007;Lund et al, 2011).…”

P21‐Activated Kinase 4 Regulates the Cyclin‐Dependent Kinase Inhibitor P57Kip2 in Human Breast Cancer

“…Flag control or Flag-PAK4 lentivirus infected MCF-7 cells were treated with 10 lM MG132 for 24 h (Adams et al, 2009). Cell lysates prepared in NP-40 lysis buffer were precleared with isotypical preimmune IgG plus Protein A/G beads, followed by a brief centrifugation at 500g.…”