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Akt phosphorylation is a major driver of cell survival, motility, and proliferation in development and disease, causing increased interest in upstream regulators of Akt like mTOR complex 2 (mTORC2). We used genetic disruption of Rictor to impair mTORC2 activity in mouse mammary epithelia, which decreased Akt phosphorylation, ductal length, secondary branching, cell motility, and cell survival. These effects were recapitulated with a pharmacological dual inhibitor of mTORC1/mTORC2, but not upon genetic disruption of mTORC1 function via Raptor deletion. Surprisingly, Akt re-activation was not sufficient to rescue cell survival or invasion, and modestly increased branching of mTORC2-impaired mammary epithelial cells (MECs) in culture and in vivo. However, another mTORC2 substrate, protein kinase C (PKC)-alpha, fully rescued mTORC2-impaired MEC branching, invasion, and survival, as well as branching morphogenesis in vivo. PKC-alpha-mediated signaling through the small GTPase Rac1 was necessary for mTORC2-dependent mammary epithelial development during puberty, revealing a novel role for Rictor/mTORC2 in MEC survival and motility during branching morphogenesis through a PKC-alpha/Rac1-dependent mechanism.
Kidney size adaptively increases as mammals grow and in response to the loss of 1 kidney. It is not clear how kidneys size themselves or if the processes that adapt kidney mass to lean body mass also mediate renal hypertrophy following unilateral nephrectomy (UNX). Here, we demonstrated that mice harboring a proximal tubule-specific deletion of Pten (Pten(ptKO)) have greatly enlarged kidneys as the result of persistent activation of the class I PI3K/mTORC2/AKT pathway and an increase of the antiproliferative signals p21(Cip1/WAF) and p27(Kip1). Administration of rapamycin to Pten(ptKO) mice diminished hypertrophy. Proximal tubule-specific deletion of Egfr in Pten(ptKO) mice also attenuated class I PI3K/mTORC2/AKT signaling and reduced the size of enlarged kidneys. In Pten(ptKO) mice, UNX further increased mTORC1 activation and hypertrophy in the remaining kidney; however, mTORC2-dependent AKT phosphorylation did not increase further in the remaining kidney of Pten(ptKO) mice, nor was it induced in the remaining kidney of WT mice. After UNX, renal blood flow and amino acid delivery to the remaining kidney rose abruptly, followed by increased amino acid content and activation of a class III PI3K/mTORC1/S6K1 pathway. Thus, our findings demonstrate context-dependent roles for EGFR-modulated class I PI3K/mTORC2/AKT signaling in the normal adaptation of kidney size and PTEN-independent, nutrient-dependent class III PI3K/mTORC1/S6K1 signaling in the compensatory enlargement of the remaining kidney following UNX.
The mTOR and Hippo pathways have recently emerged as the major signaling transduction cascades regulating organ size and cellular homeostasis. However, direct crosstalk between two pathways is yet to be determined. Here, we demonstrate that mTORC2 is a direct negative regulator of the MST1 kinase, a key component of the Hippo pathway. mTORC2 phosphorylates MST1 at serine 438 in the SARAH domain, thereby reducing its homodimerization and activity. We found that Rictor/mTORC2 preserves cardiac structure and function by restraining the activity of MST1 kinase. Cardiac-specific mTORC2 disruption through Rictor deletion leads to a marked activation of MST1 that, in turn, promotes cardiac dysfunction and dilation, impairing cardiac growth and adaptation in response to pressure overload. In conclusion, our study demonstrates the existence of a direct crosstalk between mTORC2 and MST1 that is critical for cardiac cell survival and growth.
Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates a diverse array of cellular processes, including cell growth, survival, metabolism, and cytoskeleton dynamics. mTOR functions in two distinct complexes, mTORC1 and mTORC2, whose activities and substrate specificities are regulated by complex specific cofactors, including Raptor and Rictor, respectively. Little is known regarding the relative contribution of mTORC1 versus mTORC2 in vascular endothelial cells. Using mouse models of Raptor or Rictor gene targeting, we discovered that Rictor ablation inhibited vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation and assembly in vitro and angiogenesis in vivo, whereas the loss of Raptor had only a modest effect on endothelial cells (ECs). Mechanistically, the loss of Rictor reduced the phosphorylation of AKT, protein kinase Cα (PKCα), and NDRG1 without affecting the mTORC1 pathway. In contrast, the loss of Raptor increased the phosphorylation of AKT despite inhibiting the phosphorylation of S6K1, a direct target of mTORC1. Reconstitution of Rictor-null cells with myristoylated AKT (Myr-AKT) rescued vascular assembly in Rictor-deficient endothelial cells, whereas PKCα rescued proliferation defects. Furthermore, tumor neovascularization in vivo was significantly decreased upon EC-specific Rictor deletion in mice. These data indicate that mTORC2 is a critical signaling node required for VEGF-mediated angiogenesis through the regulation of AKT and PKCα in vascular endothelial cells.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Tuberous sclerosis complex (TSC) is a multi-organ disorder caused by mutations of the TSC1 or TSC2 genes. A key function of these genes is to inhibit mTORC1 (mechanistic target of rapamycin complex 1) kinase signaling. Cells deficient for TSC1 or TSC2 have increased mTORC1 signaling and give rise to benign tumors, although, as a rule, true malignancies are rarely seen. In contrast, other disorders with increased mTOR signaling typically have overt malignancies. A better understanding of genetic mechanisms that govern the transformation of benign cells to malignant ones is crucial to understand cancer pathogenesis. We generated a zebrafish model of TSC and cancer progression by placing a heterozygous mutation of the tsc2 gene in a p53 mutant background. Unlike tsc2 heterozygous mutant zebrafish, which never exhibited cancers, compound tsc2;p53 mutants had malignant tumors in multiple organs. Tumorigenesis was enhanced compared with p53 mutant zebrafish. p53 mutants also had increased mTORC1 signaling that was further enhanced in tsc2;p53 compound mutants. We found increased expression of Hif1-α, Hif2-α and Vegf-c in tsc2;p53 compound mutant zebrafish compared with p53 mutant zebrafish. Expression of these proteins probably underlies the increased angiogenesis seen in compound mutant zebrafish compared with p53 mutants and might further drive cancer progression. Treatment of p53 and compound mutant zebrafish with the mTORC1 inhibitor rapamycin caused rapid shrinkage of tumor size and decreased caliber of tumor-associated blood vessels. This is the first report using an animal model to show interactions between tsc2, mTORC1 and p53 during tumorigenesis. These results might explain why individuals with TSC rarely have malignant tumors, but also suggest that cancer arising in individuals without TSC might be influenced by the status of TSC1 and/or TSC2 mutations and be potentially treatable with mTORC1 inhibitors.
Tuberous sclerosis complex (TSC) is a multisystem genetic disorder with severe neurologic manifestations, including epilepsy, autism, anxiety and attention deficit hyperactivity disorder. TSC is caused by the loss of either the TSC1 or TSC2 genes that normally regulate the mammalian target of rapamycin (mTOR) kinase. mTOR exists within two distinct complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Loss of either TSC gene leads to increased mTORC1 but decreased mTORC2 signaling. As the contribution of decreased mTORC2 signaling to neural development and homeostasis has not been well studied, we generated a conditional knockout (CKO) of Rictor, a key component of mTORC2. mTORC2 signaling is impaired in the brain, whereas mTORC1 signaling is unchanged. Rictor CKO mice have small brains and bodies, normal lifespan and are fertile. Cortical layering is normal, but neurons are smaller than those in control brains. Seizures were not observed, although excessive slow activity was seen on electroencephalography. Rictor CKO mice are hyperactive and have reduced anxiety-like behavior. Finally, there is decreased white matter and increased levels of monoamine neurotransmitters in the cerebral cortex. Loss of mTORC2 signaling in the cortex independent of mTORC1 can disrupt normal brain development and function and may contribute to some of the neurologic manifestations seen in TSC.