PAK2 is an effector of TSC1/2 signaling independent of mTOR and a potential therapeutic target for Tuberous Sclerosis Complex

Scientific Reports, Sep 2015

Tuberous sclerosis complex (TSC) is caused by inactivating mutations in either TSC1 or TSC2 and is characterized by uncontrolled mTORC1 activation. Drugs that reduce mTOR activity are only partially successful in the treatment of TSC, suggesting that mTOR-independent pathways play a role in disease development. Here, kinome profiles of wild-type and Tsc2−/− mouse embryonic fibroblasts (MEFs) were generated, revealing a prominent role for PAK2 in signal transduction downstream of TSC1/2. Further investigation showed that the effect of the TSC1/2 complex on PAK2 is mediated through RHEB, but is independent of mTOR and p21RAC. We also demonstrated that PAK2 over-activation is likely responsible for the migratory and cell cycle abnormalities observed in Tsc2−/− MEFs. Finally, we detected high levels of PAK2 activation in giant cells in the brains of TSC patients. These results show that PAK2 is a direct effector of TSC1-TSC2-RHEB signaling and a new target for rational drug therapy in TSC.

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PAK2 is an effector of TSC1/2 signaling independent of mTOR and a potential therapeutic target for Tuberous Sclerosis Complex

Abstract Tuberous sclerosis complex (TSC) is caused by inactivating mutations in either TSC1 or TSC2 and is characterized by uncontrolled mTORC1 activation. Drugs that reduce mTOR activity are only partially successful in the treatment of TSC, suggesting that mTOR-independent pathways play a role in disease development. Here, kinome profiles of wild-type and Tsc2−/− mouse embryonic fibroblasts (MEFs) were generated, revealing a prominent role for PAK2 in signal transduction downstream of TSC1/2. Further investigation showed that the effect of the TSC1/2 complex on PAK2 is mediated through RHEB, but is independent of mTOR and p21RAC. We also demonstrated that PAK2 over-activation is likely responsible for the migratory and cell cycle abnormalities observed in Tsc2−/− MEFs. Finally, we detected high levels of PAK2 activation in giant cells in the brains of TSC patients. These results show that PAK2 is a direct effector of TSC1-TSC2-RHEB signaling and a new target for rational drug therapy in TSC. Introduction Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder characterized by the presence of benign tumours in the brain and in other vital organs such as the kidneys, heart, eyes, lungs, and skin, and is associated with severe neurological and behavioral symptoms. Up to 90% of patients suffer from epilepsy, and almost 50% have intellectual disabilities1. TSC is caused by a heterozygous mutation in either TSC12,3 or TSC24,5 which encode for hamartin (TSC1) and tuberin (TSC2), respectively6. These two proteins interact with each other7 to form a functional heterodimeric complex that controls different aspects of cellular metabolism by its GTPase activating function towards the Ras homolog enriched in brain protein (RHEB)8. In a normal situation, the TSC1-TSC2 complex inactivates RHEB by stimulating the conversion of RHEB-GTP to RHEB-GDP, which subsequently downregulates the mammallian target of rapamycin (mTOR) signaling pathway9. In the presence of a mutation in either TSC1 or TSC2, the TSC1-TSC2 complex is unable to form and RHEB is kept in a permanently active, GTP-bound state, leading to constitutive activation of the mTOR pathway and, as a consequence, to uncontrolled cell growth. The importance of mTOR signaling downstream of the TSC genes is further illustrated by the observation that upregulation of TSC1 following ischaemia induces protective autophagia via inhibition of the mTOR pathway and can protect neurons from ischaemia-induced cell death10. mTOR is an important regulator of cellular metabolism and has multiple downstream targets including p70S6 kinase and the eukaryotic initiation factor 4E (eIF4E)-binding proteins 1 and 2 (4E-BP1 and 4E-BP2)11. Various molecules are potent inhibitors of mTOR and are used clinically to combat disease characterised by excessive mTOR activity, including TSC. Rapamycin and its analogues such as sirolimus and everolimus, play a prominent role in this respect. Rapamycin is a macrolide antibiotic that blocks mTOR signaling by forming an inhibitory complex with the cytosolic FK-binding protein-12 (FKBP-12)12. The Rapamycin-FKBP12 complex directly binds and inhibits mTOR when present in a protein complex consisting of mTOR, Raptor, LST8, PRAS and DEPTOR, together known as mTORC1, but not when present in the protein complex mTORC2 (consisting of mTOR, Rictor, LST8, mSIN1, DEPTOR and Protor). mTORC1 inhibition by rapamycin is now firmly established as a therapeutic option in various diseases, especially transplantation medicine and various forms of cancer. Indeed, in two prospective studies sirolimus proved to be safe and effective for the treatment of TSC-associated renal angiomyolipoma13,14. Furthermore, everolimus substantially and significantly reduced the volume of subependymal giant cell astrocytomas in an open-label phase 1/2 study15, while in a larger double-blind placebo-controlled phase III study, it was shown to reduce tumour size and seizure frequency in a significant fraction of patients with TSC and associated subependymal giant cell astrocytoma16. Unfortunately, some clinical manifestations of TSC, especially lung function14 and neurological symptoms, do not or only partially react to prolonged treatment with rapamycin analogues. Although none of the everolimus-treated patients progressed, only 27 (35%) of the 78 patients in the everolimus-treated group of the phase III study (versus none of the placebo group) was considered to have a response in terms of a reduction in the total volume of all subependymal giant cell astrocytomas16. Thus mTOR inhibition was only partially effective in these patients. Furthermore, TSC associated tumours are highly autophagia dependent, and as rapamycin analogues are well-known to stimulate autophagia, their administration will provide a pro-survival stimulus that will limit their own efficacy, contributing to the partial clinical response seen for human angiomyolipomas and subependymal giant cell (...truncated)


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Maria M. Alves, Gwenny M. Fuhler, Karla C.S. Queiroz, Jetse Scholma, Susan Goorden, Jasper Anink, C. Arnold Spek, Marianne Hoogeveen-Westerveld, Marco J. Bruno, Mark Nellist, Ype Elgersma, Eleonora Aronica, Maikel P. Peppelenbosch. PAK2 is an effector of TSC1/2 signaling independent of mTOR and a potential therapeutic target for Tuberous Sclerosis Complex, Scientific Reports, 2015, Issue: 5, DOI: 10.1038/srep14534