Screening health-promoting compounds for their capacity to induce the activity of FOXO3

Several chemical compounds including natural products have been suggested as being effective against age-related diseases or as beneficial for a healthy life. On the other hand, forkhead box O (FOXO) proteins are emerging as key cellular components associated with extreme human longevity. FOXO proteins are mainly regulated by posttranslational modifications and as these modifications are reversible, activation and inactivation of FOXO are attainable through pharmacological treatment. Here, we questioned whether a panel of compounds with known health-beneficial properties has the capacity to induce the activity of FOXO factors. We show that resveratrol, a phytoalexin present in grapes and other food products, the amide alkaloid piperlongumine found in the fruit of the long pepper, and the plant-derived beta-carboline compound harmine induced nuclear translocation of FOXO3. We also show that piperlongumine and harmine but not resveratrol activate FOXO-dependent transcription. We determined the half maximal effective concentration (EC50) values for resveratrol, piperlongumine, and harmine for FOXO translocation, and analyzed their inhibitory impact on chromosomal maintenance 1 (CRM1)-mediated nuclear export and the production of reactive oxygen species (ROS). We also used chemical biology approach and Western blot analysis to explore the underlying molecular mechanisms. We show that harmine, piperlongumine, and resveratrol activate FOXO3 independently of phosphoinositide 3-kinase (PI3K)/AKT signaling and the CRM1-mediated nuclear export. The effect of harmine on FOXO3 activity is at least partially mediated through the inhibition of dual-specificity tyrosine (Y) phosphorylationregulated kinase 1A (DYRK1A) and can be reverted by the inhibition of sirtuins (SIRTs).Spanish Government RTI2018-094629-B-I00, 2 LPCC-NRS/Terry Fox grants 2016/2017, 2017/2018, German Research Foundation (DFG) BR1034/6-1info:eu-repo/semantics/publishedVersio

mTORC2 Is the major second layer kinase negatively regulating FOXO3 activity

Forkhead box O (FOXO) proteins are transcription factors involved in cancer and aging and their pharmacological manipulation could be beneficial for the treatment of cancer and healthy aging. FOXO proteins are mainly regulated by post-translational modifications including phosphorylation, acetylation and ubiquitination. As these modifications are reversible, activation and inactivation of FOXO factors is attainable through pharmacological treatment. One major regulatory input of FOXO signaling is mediated by protein kinases. Here, we use specific inhibitors against different kinases including PI3K, mTOR, MEK and ALK, and other receptor tyrosine kinases (RTKs) to determine their effect on FOXO3 activity. While we show that inhibition of PI3K efficiently drives FOXO3 into the cell nucleus, the dual PI3K/mTOR inhibitors dactolisib and PI-103 induce nuclear FOXO translocation more potently than the PI3Kδ inhibitor idelalisib. Furthermore, specific inhibition of mTOR kinase activity affecting both mTORC1 and mTORC2 potently induced nuclear translocation of FOXO3, while rapamycin, which specifically inhibits the mTORC1, failed to affect FOXO3. Interestingly, inhibition of the MAPK pathway had no effect on the localization of FOXO3 and upstream RTK inhibition only weakly induced nuclear FOXO3. We also measured the effect of the test compounds on the phosphorylation status of AKT, FOXO3 and ERK, on FOXO-dependent transcriptional activity and on the subcellular localization of other FOXO isoforms. We conclude that mTORC2 is the most important second layer kinase negatively regulating FOXO activity.info:eu-repo/semantics/publishedVersio

Harmine and Piperlongumine Revert TRIB2-Mediated Drug Resistance.

Therapy resistance is responsible for most relapses in patients with cancer and is the major challenge to improving the clinical outcome. The pseudokinase Tribbles homologue 2 (TRIB2) has been characterized as an important driver of resistance to several anti-cancer drugs, including the dual ATP-competitive PI3K and mTOR inhibitor dactolisib (BEZ235). TRIB2 promotes AKT activity, leading to the inactivation of FOXO transcription factors, which are known to mediate the cell response to antitumor drugs. To characterize the downstream events of TRIB2 activity, we analyzed the gene expression profiles of isogenic cell lines with different TRIB2 statuses by RNA sequencing. Using a connectivity map-based computational approach, we identified drug-induced gene-expression profiles that invert the TRIB2-associated expression profile. In particular, the natural alkaloids harmine and piperlongumine not only produced inverse gene expression profiles but also synergistically increased BEZ235-induced cell toxicity. Importantly, both agents promote FOXO nuclear translocation without interfering with the nuclear export machinery and induce the transcription of FOXO target genes. Our results highlight the great potential of this approach for drug repurposing and suggest that harmine and piperlongumine or similar compounds might be useful in the clinic to overcome TRIB2-mediated therapy resistance in cancer patients.This work was supported by the FUNDAÇÃO PARA A CIÊNCIA E TECNOLOGIA (FCT) Research Center Grant UID/BIM/04773/2013, Centre for Biomedical Research (CBMR), and by the Spanish Ministry of Science, Innovation and Universities through Grant RTI2018-094629-B-I00 to WL. B.I.F. was supported by FCT-SFRH/BPD/100434/2014 and the Marie Curie Individual Fellowship project TRIBBLES (#748585). This work was also supported by two LPCC-NRS/Terry Fox grants (2016/2017; 2017/2018). S. Machado is the recipient of a ProRegeM grant PD/BD/114258/2016. I. Duarte was supported by a scholarship from FCT Grant PTDC/BEX-BID/5410/2014S

Multiplexed cellular profiling identifies an organoselenium compound as an inhibitor of CRM1-mediated nuclear export

Chromosomal region maintenance 1 (CRM1 also known as Xpo1 and exportin-1) is the receptor for the nuclear export controlling the intracellular localization and function of many cellular and viral proteins that play a crucial role in viral infections and cancer. The inhibition of CRM1 has emerged as a promising therapeutic approach to interfere with the lifecycle of many viruses, for the treatment of cancer, and to overcome therapy resistance. Recently, selinexor has been approved as the first CRM1 inhibitor for the treatment of multiple myeloma, providing proof of concept for this therapeutic option with a new mode of action. However, selinexor is associated with dose-limiting toxicity and hence, the discovery of alternative small molecule leads that could be developed as less toxic anticancer and antiviral therapeutics will have a significant impact in the clinic. Here, we report a CRM1 inhibitor discovery platform. The development of this platform includes reporter cell lines that monitor CRM1 activity by using red fluorescent protein or green fluorescent protein-labeled HIV-1 Rev protein with a strong heterologous nuclear export signal. Simultaneously, the intracellular localization of other proteins, to be interrogated for their capacity to undergo CRM1-mediated export, can be followed by co-culturing stable cell lines expressing fluorescent fusion proteins. We used this platform to interrogate the mode of nuclear export of several proteins, including PDK1, p110α, STAT5A, FOXO1, 3, 4 and TRIB2, and to screen a compound collection. We show that while p110α partially relies on CRM1-dependent nuclear export, TRIB2 is exported from the nucleus in a CRM1-independent manner. Compound screening revealed the striking activity of an organoselenium compound on the CRM1 nuclear export receptorThis article is based upon work from COST Action STRATAGEM, CA17104, supported by COST (European Cooperation in Science and Technology) (www.cost.eu, accessed in March 2022). Romano Silvestri is indebted to AIRC, IG 2020, code no. 24703. This work was supported by the Spanish Ministry of Science, Innovation and Universities through Grant RTI2018-094629-B-I00 to Wolfgang Link. Miguel Machuqueiro thanks Fundaçao para a Ciência e Tecnologia ˜ (Portugal) for CEECIND/02300/2017 (grant), UIDB/04046/2020 and UIDP/04046/2020 (projects

Papel de los dominios adaptadores de c-Src en cáncer de mama

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 21-02-2019Esta tesis tiene embargado el acceso al texto completo hasta el 21-08-2020c-Src es el prototipo de la familia de proteínas tirosinas quinasas no receptoras Src. Su sobreexpresión e hiperactivación se ha asociado a múltiples tipos de tumores. En cáncer de mama se ha descrito el papel de su actividad quinasa en la progresión tumoral. Sin embargo, c-Src también posee los dominios adaptadores SH2 y SH3, que son muy importantes en la interacción con otras proteínas para regular procesos como la migración. Esta Tesis Doctoral se centra en el estudio de estos dominios en el cáncer mamario humano, con el fin de conocer el papel que juegan en diferentes procesos tumorales. Para ello se han expresado de manera condicional diferentes mutantes de c-Src en células de cáncer de mama humano. En células MCF-7 se ha comprobado que la expresión de SrcDN, mutante de c-Src sin actividad quinasa y con los dominios adaptadores siempre expuestos, reduce su tumorigénicidad in vivo, afectando a la capacidad de autorrenovación de sus células troncales tumorales (CSCs), las responsables de la tumorigénesis. Esto se debe, al menos en parte, a la reducción de la glicólisis anaeróbica o fenotipo Warburg producido por la expresión de SrcDN en sus CSCs. En células de cáncer mamario triple negativo (TNBC) MDA-MB-231 y SUM159PT, procedentes de tumores humanos muy agresivos, la expresión de SrcDN también redujo la capacidad de autorrenovación de CSCs. La expresión de mutantes sin funcionalidad en alguno o en ambos dominios adaptadores disminuyó igualmente la capacidad de autorrenovación de CSCs, poniendo de manifiesto la importancia de los dominios adaptadores SH2 y SH3 de c-Src en la carcinogénesis mamaria. Además, el estudio de otros procesos asociados a la tumorigénesis como el crecimiento independiente de anclaje, proliferación, migración e invasividad, mostraron que el dominio adaptador SH2 juega un papel muy importante en todos ellos. Por tanto, el dominio SH2 de c-Src puede ser una buena diana terapéutica, que en combinación con inhibidores de la actividad quinasa de c-Src produzcan mejores resultados curativos en pacientes con TNBC, para los que actualmente el pronóstico es muy negativo.c-Src is the prototype of the family of non-receptor protein tyrosine kinases Src. Its overexpression and hyperactivation has been associated with multiple types of tumours. In breast cancer, the role of its kinase activity in tumour progression has been described. However, c-Src also has the adapter domains SH2 and SH3, which are very important for the interaction with other cellular proteins to regulate processes such as migration. This Doctoral Thesis focuses on the study of these domains in human breast cancer, in order to understand the role they play in different tumour processes. For this reason, different mutants of c-Src have been conditionally expressed in human breast cancer cells. In MCF-7 cells, expression of SrcDN, a mutant of c-Src without kinase activity and with the adapter domains always exposed, reduces its tumorigenicity in vivo, affecting the self-renewal capacity of its tumour stem cells (CSCs), responsible for tumorigenesis. This is due, at least in part, to the reduction of anaerobic glycolysis or Warburg phenotype produced by the expression of SrcDN in their CSCs. In the triple negative breast cancer (TNBC) cells MDA-MB-231 and SUM159PT, from very aggressive human tumours, the expression of SrcDN also reduced the self-renewal capacity of CSCs. The expression of mutants without functionality in either or both of the adapter domains also decreased the self-renewal capacity of CSCs, demonstrating the importance of the SH2 and SH3 adapter domains of c-Src in mammary gland carcinogenesis. In addition, the study of other processes associated with tumorigenesis such as anchorage independent growth, proliferation, migration and invasiveness, showed that the SH2 adapter domain of c-Src plays a very important role in all of them. Therefore, the SH2 domain of c-Src can be a good therapeutic target, which in combination with inhibitors of c-Src kinase activity produce better curative results in patients with TNBC, for which the prognosis is currently very negative