Abstract
FOXO proteins are transcription factors with important roles in the regulation of the expression of genes involved in cell growth, proliferation, differentiation, and longevity. FOXO proteins are active in the nucleus but, upon post-translational modification they form a docking site for 14-3-3 proteins and are translocated to the cytoplasm where they are inactive.
We make use of this regulatory mechanism of FOXO proteins to develop an image-based high-throughput screening platform to detect compounds that regulate FOXO3 subcellular localization. This system has proven a powerful tool to isolate inhibitors of proteins upstream of FOXO, such as PI3K inhibitors.
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References
Link W, Fernandez-Marcos PJ (2017) FOXO transcription factors at the interface of metabolism and cancer. Int J Cancer 141:2379–2391. https://doi.org/10.1002/ijc.30840
Mawal-Dewan M, Lorenzini A, Frisoni L et al (2002) Regulation of collagenase expression during replicative senescence in human fibroblasts by Akt-forkhead signaling. J Biol Chem 277:7857–7864. https://doi.org/10.1074/jbc.M104515200
Brunet A, Bonni A, Zigmond MJ et al (1999) Akt promotes cell survival by phosphorylating and inhibiting a forkhead transcription factor. Cell 96:857–868. https://doi.org/10.1016/S0092-8674(00)80595-4
Zanella F, dos Santos NR, Link W (2013) Moving to the core: spatiotemporal analysis of forkhead box O (FOXO) and nuclear factor-κB (NF-κB) nuclear translocation. Traffic 14:247–258. https://doi.org/10.1111/tra.12034
Hung M-C, Link W (2011) Protein localization in disease and therapy. J Cell Sci 124:3381–3392. https://doi.org/10.1242/jcs.089110
Hill R, Cautain B, de Pedro N, Link W (2014) Targeting nucleocytoplasmic transport in cancer therapy. Oncotarget 5:11–28. https://doi.org/10.18632/oncotarget.1457
Zanella F, Rosado A, García B et al (2008) Chemical genetic analysis of FOXO nuclear-cytoplasmic shuttling by using image-based cell screening. Chembiochem 9:2229–2237. https://doi.org/10.1002/cbic.200800255
Zanella F, Rosado A, Garcia B et al (2009) Using multiplexed regulation of luciferase activity and GFP translocation to screen for FOXO modulators. BMC Cell Biol 10:14. https://doi.org/10.1186/1471-2121-10-14
Zanella F, Rosado A, Blanco F et al (2007) An HTS approach to screen for antagonists of the nuclear export machinery using high content cell-based assays. Assay Drug Dev Technol 5:333–341. https://doi.org/10.1089/adt.2007.058
Link W, Oyarzabal J, Serelde BG et al (2009) Chemical interrogation of FOXO3a nuclear translocation identifies potent and selective inhibitors of phosphoinositide 3-kinases. J Biol Chem 284:28392–28400. https://doi.org/10.1074/jbc.M109.038984
Zanella F, Lorens JB, Link W (2010) High content screening: seeing is believing. Trends Biotechnol 28:237–245. https://doi.org/10.1016/j.tibtech.2010.02.005
Hill R, Kalathur RKR, Callejas S et al (2014) A novel phosphatidylinositol 3-kinase (PI3K) inhibitor directs a potent FOXO-dependent, p53-independent cell cycle arrest phenotype characterized by the differential induction of a subset of FOXO-regulated genes. Breast Cancer Res 16:482. https://doi.org/10.1186/s13058-014-0482-y
Lopez-Guadamillas E, Muñoz-Martin M, Martinez S et al (2016) PI3Kα inhibition reduces obesity in mice. Aging (Albany NY) 8:2747–2753. https://doi.org/10.18632/aging.101075
Ortega-Molina A, Lopez-Guadamillas E, Mattison JA et al (2015) Pharmacological inhibition of PI3K reduces adiposity and metabolic syndrome in obese mice and rhesus monkeys. Cell Metab 21:558–570. https://doi.org/10.1016/j.cmet.2015.02.017
Zhang J-H, Chung O (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4:67–73. https://doi.org/10.1177/108705719900400206
Kozak K (2009) Data mining techniques in high content screening: a survey. J Comput Sci Syst Biol 2:219–239. https://doi.org/10.4172/jcsb.1000035
Acknowledgments
We thank Lola Martinez for her assistance in Flow Cytometry. Work in the laboratory of P.J.F.-M. was funded by the IMDEA Food, the Spanish Association Against Cancer (aecc) and the FBBVA and Ramon Areces Foundations. This work was supported by Fundação para a Ciência e a Tecnologia (FCT) Research Center Grant UID/BIM/04773/2013 Centre for Biomedical Research 1334. Work in the laboratory of WL was funded by a research grant from Liga Portuguesa Contra o Cancro – Núcleo Regional do Sul (LPCC/NRS), Terry Fox.
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Barradas, M., Link, W., Megias, D., Fernandez-Marcos, P.J. (2019). High-Throughput Image-Based Screening to Identify Chemical Compounds Capable of Activating FOXO. In: Link, W. (eds) FOXO Transcription Factors. Methods in Molecular Biology, vol 1890. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-8900-3_13
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DOI: https://doi.org/10.1007/978-1-4939-8900-3_13
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