FOXO4 regulates metastasis by binding to and suppressing RUNX2 transactivation ability.

Abstract

<p><b>A.</b> Promoter regions of <i>RUNX2, PIP, PGC, PLA2G16</i> and <i>CAMK2N1</i> showing potential FOXO (DBE) and RUNX2 (RBS) binding sites relative to first exons. <b>B.</b> Matrigel invasion assay of LNCaP cells expressing control, FOXO4 or FOXO4 plus RUNX2 shRNAs. Error bars, S.E. of triplicate experiments. **, P<0.02. <b>C.</b> Relative RUNX2 RNA levels, as assessed by qRT-PCR in control shRNA vs. shFOXO4 LNCaP cells, primary tumors or LN metastases. RNA levels in each control condition were set to 1. Error bars, S.E. of triplicate experiments. n.s., not significant. Lysates of HEK293T cells transfected with HA-RUNX2 and Myc-FOXO4 were either analyzed by IB for HA, Myc or GAPDH, or immunoprecipitated with anti-myc and blotted with anti-HA (<b>D</b>), or immunoprecipitated with anti-HA and blotted with anti-Myc (<b>E</b>). <b>F.</b> Chromatin from LNCaP[vector] (control) or LNCaP[shFOXO4] cells were immunoprecipitated with control IgG or RUNX2 Ab, and the precipitated DNA subjected to qPCR using PIP promoter primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101411#pone.0101411.s007" target="_blank">Table S2</a>). Error bars, S.E. of triplicates. **, P<0.01. <b>G.</b> Chromatin from HEK293T cells transfected with expression plasmids for RUNX2, RUNX2+FOXO4 or empty vector were immunoprecipitated with control IgG or HA Ab, then analyzed for PIP DNA by qPCR as in <b>F</b>.</p