The Tribbles pseudokinases family consists of TRIB1, TRIB2, TRIB3 and STK40 and, although evolutionarily conserved, they have distinctive characteristics. Tribbles members are expressed in a context and cell compartment-dependent manner. For example, TRIB1 and TRIB2 have potent oncogenic activities in vertebrate cells. Since the identification of Tribbles proteins as modulators of multiple signalling pathways, recent studies have linked their expression with several pathologies, including cancer. Tribbles proteins act as protein adaptors involved in the ubiquitin-proteasome degradation system, as they bridge the gap between substrates and E3 ligases. Between TRIB family members, TRIB2 is the most ancestral member of the family. TRIB2 is involved in protein homeostasis regulation of C/EBPα, β-catenin and TCF4. On the other hand, TRIB2 interacts with MAPKK, AKT and NFkB proteins, involved in cell survival, proliferation and immune response. Here, we review the characteristic features of TRIB2 structure and signalling and its role in many cancer subtypes with an emphasis on TRIB2 function in therapy resistance in melanoma, leukemia and glioblastoma. The strong evidence between TRIB2 expression and chemoresistance provides an attractive opportunity for targeting TRIB2.

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 β-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).

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.

Tribbles homolog 2 (TRIB2) is one of three members of the Tribbles family of pseudo serine/threonine kinases. It acts as an oncogene whose expression is correlated with tumor progression and therapy resistance in melanoma. TRIB2 has been implicated in conferring resistance to various anti-cancer therapies suggesting TRIB2 as a therapeutic target for resistant tumors. This study explores the pharmacological targeting of TRIB2, revealing several independent routes of pharmacological manipulations. TRIB2 is a short-lived protein stabilized by inhibition of the PI3K/AKT pathway. Conversely, inhibitors of BRAF, MEK and ERK significantly decrease TRIB2 expression by a mechanism that involves transcription. Additionally, Polo-Like Kinases (PLKs) inhibitors significantly reduce TRIB2 protein expression and stability. Strikingly, increasing concentrations of the kinase inhibitor PIK-75 effectively eliminate TRIB2 in melanoma cells surpassing its PI3K inhibitory activity suggesting the destabilizing effect of the inhibition of an unknown kinase. TMT-based proteomics, chemical biology and genetic manipulation revealed CDK12 as the kinase that mediates the destabilizing effect on TRIB2. Overall, we identify three distinct classes of compounds that efficiently eliminate the oncogenic TRIB2 protein from melanoma cells based on different molecular mechanisms and exhibiting 40 to 200 times greater potency than the previously reported afatinib.