Chemical Genetic Analysis of FOXO Nuclear–Cytoplasmic Shuttling by Using Image‐Based Cell Screening

Abstract

FOXO proteins are direct targets of PI3K/Akt signaling and they integrate the signals of several other transduction pathways at the transcriptional level. FOXO transcription factors are involved in normal cell homeostasis and neoplasia, and they are regulated by multiple post-transcriptional modifications. In cancer research, the regulation of the FOXO factors is receiving increasing attention as their activation has been linked to cell-cycle arrest and apoptosis. Hence, FOXO proteins have been proposed to act as tumor suppressors. Here, we applied a chemical biology approach to study the mechanisms that influence the intracellular localization of the FOXO family member FOXO3a. We established a high-throughput cellular-imaging assay that monitors the nuclear-cytoplasmic translocation of a GFP-FOXO3a fusion protein in tumor cells. Nuclear accumulation of fluorescent signals upon treatment with the known PI3K inhibitors LY294002, wortmannin, PIK-75, and PI-103 was dose dependent and agreed well with the IC(50) values reported for PI3Kalpha inhibition in vitro. Additionally, we identified 17 compounds from a panel of 73 low-molecular-weight compounds capable of inducing the nuclear accumulation of GFP-FOXO. These compounds include chemicals known to interfere with components of the PI3K/Akt signaling pathway, as well as with nuclear export and Ca(2+)/calmodulin (CaM)-dependent signaling events. Interestingly, the therapeutic agent vinblastine induced efficient nuclear translocation of the FOXO reporter protein. Our data illustrate the potential of chemical genetics when combined with robust and sensitive high-content-screening technology.

Chemical Interrogation of FOXO3a Nuclear Translocation Identifies Potent and Selective Inhibitors of Phosphoinositide 3-Kinases

“…This was followed by molecular modeling and traditional medicinal chemistry that led to the discovery of ETP-45658 a potent and selective inhibitor of PI3 kinases. The primary screen of 33,992 compounds followed the intracellular location of the Akt effector, FOXO3a, in tumor cells (16). Compounds found that induced the nuclear translocation of GFP-FOXO yet did not affect general nuclear export were profiled against a small panel of protein kinases involved in the regulation of FOXO nuclear localization.…”

“…U2foxRELOC and U2nesRELOC Assay-The U2nesRELOC assay and the U2foxRELOC assay have been described previously (16,19). Briefly, cells were seeded at a density of 1.0 ϫ 10 5 cells/ml into black wall clear bottom 96-well microplates (BD Biosciences) after 12 h of incubation at 37°C with 5% CO 2 , 2 l of each test compound were transferred from the mother plates to the assay plates.…”

“…1A. As the primary filter, we used a cellular imaging assay that follows the intracellular location of FOXO proteins (16). U2OS osteosarcoma cells that stably express a green fluorescent protein (GFP)-tagged version of FOXO3a were seeded in 96-well assay plates and treated with compounds at a final concentration of 10 M for 1 h. A total of 33,992 compounds were screened for their ability to induce the translocation the GFP-FOXO3a reporter protein from the cytoplasm into the nucleus.…”

“…It is well established that FOXO is negatively regulated by various proliferative and antiapoptotic signaling pathways that activate the PI3K/Akt signaling cascade (11). Therefore, we chose to employ a high content imaging approach to monitor the nucleocytoplasmic translocation of a GFP-FOXO3a fusion protein in U2OS cells (U2foxRELOC) (16,17) as the readout for biological inhibition of PI3K/Akt signaling. The rapid kinetics of the assay allowed us to reduce the incubation time and minimize possible toxic effects that might interfere with the analysis.…”

Getting the whole picture: combining throughput with content in microscopy

“…The analysis software usually contains a set of readymade solutions for standard applications of the high-content imager such as quantifying the translocation of proteins from the cytoplasm to the nucleus (Borchert et al, 2005;Dull et al, 2010;Granas et al, 2006;Kau et al, 2003;Link et al, 2009;Straschewski et al, 2010;Xu et al, 2008;Zanella et al, 2007;Zanella et al, 2008). The combination of high-content imagers and integrated analysis programs, has contributed to many applications of HCS in both basic biology and in drug discovery (reviewed by Dragunow, 2008;Bullen, 2008).…”