Chemical genetic analysis of FOXO nuclear-cytoplasmic shuttling by using image-based cell screening
Fabian Zanella1, Aranzazú Rosado, Beatriz García
1Experimental Therapeutics Program, Centro Nacional de Investigaciones Oncologicas (CNIO), Melchor Fernandez Almagro 3, 28029 Madrid, Spain.
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.
Insights
We identified compounds that move FOXO3a protein into the nucleus, suggesting potential cancer therapies. This nuclear localization is linked to cell-cycle arrest and apoptosis, key for tumor suppression.
Area of Science:
- Molecular Biology
- Cancer Research
- Chemical Biology
Background:
- FOXO proteins are key transcription factors in cell homeostasis and cancer, regulated by PI3K/Akt signaling and post-transcriptional modifications.
- FOXO activation induces cell-cycle arrest and apoptosis, positioning FOXO proteins as potential tumor suppressors.
Purpose of the Study:
- To investigate mechanisms influencing the intracellular localization of FOXO3a using a chemical biology approach.
- To establish a high-throughput cellular-imaging assay for monitoring FOXO3a nuclear-cytoplasmic translocation in tumor cells.
Main Methods:
- Developed a high-throughput cellular-imaging assay to track GFP-FOXO3a fusion protein translocation.
- Screened 73 low-molecular-weight compounds to identify those inducing nuclear accumulation of GFP-FOXO3a.
- Validated known PI3K inhibitors (LY294002, wortmannin, PIK-75, PI-103) for their dose-dependent effect on nuclear translocation.
Main Results:
- Nuclear accumulation of GFP-FOXO3a was observed in a dose-dependent manner with known PI3K inhibitors.
- Identified 17 compounds that induce nuclear accumulation of GFP-FOXO3a, including modulators of PI3K/Akt, nuclear export, and Ca(2+)/calmodulin signaling.
- The therapeutic agent vinblastine demonstrated efficient nuclear translocation of the FOXO reporter protein.
Conclusions:
- The study highlights the potential of chemical genetics and high-content screening for dissecting signaling pathways regulating FOXO proteins.
- Identified novel compounds that promote FOXO3a nuclear localization, offering potential therapeutic strategies for cancer treatment.
- FOXO3a nuclear translocation is a sensitive readout for compounds targeting PI3K/Akt and related signaling pathways.
