Chemical genetic analysis of FOXO nuclear-cytoplasmic shuttling by using image-based cell screening.
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.
Zanella
Fabian
F
Experimental Therapeutics Program, Centro Nacional de Investigaciones Oncologicas (CNIO), Melchor Fernandez Almagro 3, 28029 Madrid, Spain.
Rosado
Aranzazú
A
García
Beatriz
B
Carnero
Amancio
A
Link
Wolfgang
W