The mTOR Deficiency in Monocytic Myeloid-Derived Suppressor Cells Protects Mouse Cardiac Allografts by Inducing Allograft Tolerance.

Background: Myeloid-derived suppressor cells (MDSCs) can prevent allograft rejection and induce immune tolerance in transplantation models. Previous studies have demonstrated that inhibition of mTOR signaling can enhance the MDSC protective effect in heart transplantation (HTx) by promoting MDSC expansion. In addition, mTOR inhibition is related to autophagy. The present study investigated the protective mechanism of mTOR-deficient monocytic MDSCs (M-MDSCs) in mouse HTx. Methods: Myeloid-specific mTOR conditional knockout mice were generated to obtain mTOR-/- M-MDSCs. The proliferation and immunosuppressive function of mTOR-/- M-MDSCs were determined by flow cytometry and T cell proliferation assays. The mTOR-/- M-MDSC intracellular autophagy levels were determined using western blotting and electron microscopy. RNAseq analysis was performed for wild-type (WT) and mTOR-/- M-MDSCs. Allogeneic HTx mouse model was established and treated with WT or mTOR-/- M-MDSCs. Enzyme-linked immunosorbent assay, flow cytometry, and immunohistochemistry assays were performed to determine WT and mTOR-/- M-MDSC-induced immune tolerance. Results: The mTOR deficiency promoted M-MDSC differentiation and enhanced intracellular autophagy levels in vivo and in vitro. mTOR deficiency also enhanced the immunosuppressive function of M-MDSCs. In addition, infusing with WT and mTOR-/- M-MDSCs prolonged cardiac allograft survival and established immune tolerance in recipient mice by inhibiting T cell activation and inducing regulatory T cells. Conclusion: mTOR deficiency enhances the immunosuppressive function of M-MDSCs and prolongs mouse cardiac allograft survival.

Cellular metabolism constrains innate immune responses in early human ontogeny.

Pathogen immune responses are profoundly attenuated in fetuses and premature infants, yet the mechanisms underlying this developmental immaturity remain unclear. Here we show transcriptomic, metabolic and polysome profiling and find that monocytes isolated from infants born early in gestation display perturbations in PPAR-γ-regulated metabolic pathways, limited glycolytic capacity and reduced ribosomal activity. These metabolic changes are linked to a lack of translation of most cytokines and of MALT1 signalosome genes essential to respond to the neonatal pathogen Candida. In contrast, they have little impact on house-keeping phagocytosis functions. Transcriptome analyses further indicate a role for mTOR and its putative negative regulator DNA Damage Inducible Transcript 4-Like in regulating these metabolic constraints. Our results provide a molecular basis for the broad susceptibility to multiple pathogens in these infants, and suggest that the fetal immune system is metabolically programmed to avoid energetically costly, dispensable and potentially harmful immune responses during ontogeny.

Adipocyte-specific DKO of Lkb1 and mTOR protects mice against HFD-induced obesity, but results in insulin resistance.

Liver kinase B1 (Lkb1) and mammalian target of rapamycin (mTOR) are key regulators of energy metabolism and cell growth. We have previously reported that adipocyte-specific KO of Lkb1 or mTOR in mice results in distinct developmental and metabolic phenotypes. Here, we aimed to assess how genetic KO of both Lkb1 and mTOR affects adipose tissue development and function in energy homeostasis. We used Adiponectin-Cre to drive adipocyte-specific double KO (DKO) of Lkb1 and mTOR in mice. We performed indirect calorimetry, glucose and insulin tolerance tests, and gene expression assays on the DKO and WT mice. We found that DKO of Lkb1 and mTOR results in reductions of brown adipose tissue and inguinal white adipose tissue mass, but in increases of liver mass. Notably, the DKO mice developed fatty liver and insulin resistance, but displayed improved glucose tolerance after high-fat diet (HFD)-feeding. Interestingly, the DKO mice were protected from HFD-induced obesity due to their higher energy expenditure and lower expression levels of adipogenic genes (CCAAT/enhancer binding protein α and PPARγ) compared with WT mice. These results together indicate that, compared with Lkb1 or mTOR single KOs, Lkb1/mTOR DKO in adipocytes results in overlapping and distinct metabolic phenotypes, and mTOR KO largely overrides the effect of Lkb1 KO.

mTOR/Raptor signaling is critical for skeletogenesis in mice through the regulation of Runx2 expression.

The mammalian target of rapamycin (mTOR)/regulatory-associated protein of mTOR (Raptor) pathway transmits and integrates different signals including growth factors, nutrients, and energy metabolism. Nearly all these signals have been found to play roles in skeletal biology. However, the contribution of mTOR/Raptor to osteoblast biology in vivo remains to be elucidated as the conclusions of recent studies are controversial. Here we report that mice with a deficiency of either mTOR or Raptor in preosteoblasts exhibited clavicular hypoplasia and delayed fontanelle fusion, similar to those found in human patients with cleidocranial dysplasia (CCD) haploinsufficient for the transcription factor runt-related transcription factor 2 (Runx2) or those identified in Runx2+/- mice. Mechanistic analysis revealed that the mTOR-Raptor-S6K1 axis regulates Runx2 expression through phosphorylation of estrogen receptor α, which binds to Distal-less homeobox 5 (DLX5) and augments the activity of Runx2 enhancer. Moreover, heterozygous mutation of raptor in osteoblasts aggravates the bone defects observed in Runx2+/- mice, indicating a genetic interaction between Raptor and Runx2. Collectively, these findings reveal that mTOR/Raptor signaling is essential for bone formation in vivo through the regulation of Runx2 expression. These results also suggest that a selective mTOR/Raptor antagonist, which has been developed for treatment of many diseases, may have the side effect of causing bone loss.

Mammalian target of rapamycin complex 1 signalling is essential for germinal centre reaction.

The mammalian target of rapamycin (mTOR) is a serine-threonine kinase that has been shown to be essential for the differentiation and function of various immune cells. Earlier in vitro studies showed that mTOR signalling regulates B-cell biology by supporting their activation and proliferation. However, how mTOR signalling temporally regulates in vivo germinal centre B (GCB) cell development and differentiation into short-lived plasma cells, long-lived plasma cells and memory cells is still not well understood. In this study, we used a combined conditional/inducible knock-out system to investigate the temporal regulation of mTOR complex 1 (mTORC1) in the GCB cell response to acute lymphocytic choriomeningitis virus infection by deleting Raptor, a main component of mTORC1, specifically in B cells in pre- and late GC phase. Early Raptor deficiency strongly inhibited GCB cell proliferation and differentiation and plasma cell differentiation. Nevertheless, late GC Raptor deficiency caused only decreases in the size of memory B cells and long-lived plasma cells through poor maintenance of GCB cells, but it did not change their differentiation. Collectively, our data revealed that mTORC1 signalling supports GCB cell responses at both early and late GC phases during viral infection but does not regulate GCB cell differentiation into memory B cells and plasma cells at the late GC stage.

mTORC1 Signaling Promotes Limb Bud Cell Growth and Chondrogenesis.

mTORC1 signaling has been shown to promote limb skeletal growth through stimulation of protein synthesis in chondrocytes. However, potential roles of mTORC1 in prechondrogenic mesenchyme have not been explored. In this study, we first deleted Raptor, a unique and essential component of mTORC1, in prechondrogenic limb mesenchymal cells. Deletion of Raptor reduced the size of limb bud cells, resulting in overall diminution of the limb bud without affecting skeletal patterning. We then examined the potential role of mTORC1 in chondrogenic differentiation in vitro. Both pharmacological and genetic disruption of mTORC1 significantly suppressed the number and size of cartilage nodules in micromass cultures of limb bud mesenchymal cells. Similarly, inhibition of mTORC1 signaling in chondrogenic ATDC5 cells greatly impaired cartilage nodule formation, and decreased the expression of the master transcriptional factor Sox9, along with the cartilage matrix genes Acan and Col2a1. Thus, we have identified an important role for mTORC1 signaling in promoting limb mesenchymal cell growth and chondrogenesis during embryonic development. J. Cell. Biochem. 118: 748-753, 2017. © 2016 Wiley Periodicals, Inc.

Deletion of mTOR in Reactive Astrocytes Suppresses Chronic Seizures in a Mouse Model of Temporal Lobe Epilepsy.

Germline and somatic mutations in key genes of the mammalian target of rapamycin (mTOR) pathway have been identified in seizure-associated disorders. mTOR mutations lead to aberrant activation of mTOR signaling, and, although affected neurons are critical for epileptogenesis, the role of mTOR activation in glial cells remains poorly understood. We previously reported a consistent activation of the mTOR pathway in astrocytes in the epileptic foci of temporal lobe epilepsy. In this study, it was demonstrated that mTOR deletion from reactive astrocytes prevents increases in seizure frequency over the disease course. By using a tamoxifen-inducible mTOR conditional knockout system and kainic acid, a model was developed that allowed astrocyte-specific mTOR gene deletion in mice with chronic epilepsy. Animals in which mTOR was deleted from 44 % of the astrocyte population exhibited a lower seizure frequency compared with controls. Down-regulation of mTOR significantly ameliorated astrogliosis in the sclerotic hippocampus but did not rescue mossy fiber sprouting. In cultured astrocytes, the mTOR pathway modulated the stability of the astroglial glutamate transporter 1 (Glt1) and influenced the ability of astrocytes to remove extracellular glutamate. Taken together, these data indicate that astrocytes with activated mTOR signaling may provide conditions that are favorable for spontaneous recurrent seizures.

Conditional Disruption of Raptor Reveals an Essential Role for mTORC1 in B Cell Development, Survival, and Metabolism.

Mechanistic target of rapamycin (mTOR) is a serine-threonine kinase that coordinates nutrient and growth factor availability with cellular growth, division, and differentiation. Studies examining the roles of mTOR signaling in immune function revealed critical roles for mTOR in regulating T cell differentiation and function. However, few studies have investigated the roles of mTOR in early B cell development. In this study, we found that mTOR is highly activated during the pro- and pre-B stages of mouse B cell development. Conditional disruption of the mTOR coactivating protein Raptor in developing mouse B cells resulted in a developmental block at the pre-B cell stage, with a corresponding lack of peripheral B cells and loss of Ag-specific Ab production. Pre-B cell survival and proliferation were significantly reduced in Raptor-deficient mice. Forced expression of a transgenic BCR or a BclxL transgene on Raptor-deficient B cells failed to rescue B cell development, suggesting that pre-BCR signaling and B cell survival are impaired in a BclxL-independent manner. Raptor-deficient pre-B cells exhibited significant decreases in oxidative phosphorylation and glycolysis, indicating that loss of mTOR signaling in B cells significantly impairs cellular metabolic capacity. Treatment of mice with rapamycin, an allosteric inhibitor of mTOR, recapitulated the early B cell developmental block. Collectively, our data reveal a previously uncharacterized role for mTOR signaling in early B cell development, survival, and metabolism.

mTOR masters monocytic myeloid-derived suppressor cells in mice with allografts or tumors.

CD11b(+) Gr1(+) myeloid-derived suppressor cells (MDSCs) play critical roles in controlling the processes of tumors, infections, autoimmunity and graft rejection. Immunosuppressive drug rapamycin (RPM), targeting on the key cellular metabolism molecule mTOR, is currently used in clinics to treat patients with allo-grafts, autoimmune diseases and tumors. However, the effect of RPM on MDSCs has not been studied. RPM significantly decreases the cell number and the immunosuppressive ability on T cells of CD11b(+) Ly6C(high) monocytic MDSCs (M-MDSCs) in both allo-grafts-transplanted and tumor-bearing mice respectively. Mice with a myeloid-specific deletion of mTOR have poor M-MDSCs after grafting with allo-skin tissue or a tumor. Grafting of allo-skin or tumors significantly activates glycolysis pathways in myeloid precursor cells in bone marrow, which is inhibited by RPM or mTOR deletion. 2-deoxyglucose (2-DG), an inhibitor of the glycolytic pathway, inhibits M-MDSC differentiation from precursors, while enhancing glycolysis by metformin significantly rescues the RPM-caused deficiency of M-MDSCs. Therefore, we offer evidence supporting that mTOR is an intrinsic factor essential for the differentiation and immunosuppressive function of M-MDSCs and that these metabolism-relevant medicines may impact MDSCs-mediated immunosuppression or immune tolerance induction, which is of considerable clinical importance in treating graft rejection, autoimmune diseases and cancers.

mTOR is necessary for proper satellite cell activity and skeletal muscle regeneration.

The serine/threonine kinase mammalian target of rapamycin (mTOR) is a key regulator of protein synthesis, cell proliferation and energy metabolism. As constitutive deletion of Mtor gene results in embryonic lethality, the function of mTOR in muscle stem cells (satellite cells) and skeletal muscle regeneration remains to be determined. In this study, we established a satellite cell specific Mtor conditional knockout (cKO) mouse model by crossing Pax7(CreER) and Mtor(flox/flox) mice. Skeletal muscle regeneration after injury was severely compromised in the absence of Mtor, indicated by increased number of necrotic myofibers infiltrated by Evans blue dye, and reduced number and size of regenerated myofibers in the Mtor cKO mice compared to wild type (WT) littermates. To dissect the cellular mechanism, we analyzed satellite cell-derived primary myoblasts grown on single myofibers or adhered to culture plates. The Mtor cKO myoblasts exhibited defective proliferation and differentiation kinetics when compared to myoblasts derived from WT littermates. At the mRNA and protein levels, the Mtor cKO myoblasts expressed lower levels of key myogenic determinant genes Pax7, Myf5, Myod, Myog than did the WT myoblasts. These results suggest that mTOR is essential for satellite cell function and skeletal muscle regeneration through controlling the expression of myogenic genes.

mTORC1 and mTORC2 selectively regulate CD8⁺ T cell differentiation.

Activation of mTOR-dependent pathways regulates the specification and differentiation of CD4+ T effector cell subsets. Herein, we show that mTOR complex 1 (mTORC1) and mTORC2 have distinct roles in the generation of CD8+ T cell effector and memory populations. Evaluation of mice with a T cell-specific deletion of the gene encoding the negative regulator of mTORC1, tuberous sclerosis complex 2 (TSC2), resulted in the generation of highly glycolytic and potent effector CD8+ T cells; however, due to constitutive mTORC1 activation, these cells retained a terminally differentiated effector phenotype and were incapable of transitioning into a memory state. In contrast, CD8+ T cells deficient in mTORC1 activity due to loss of RAS homolog enriched in brain (RHEB) failed to differentiate into effector cells but retained memory characteristics, such as surface marker expression, a lower metabolic rate, and increased longevity. However, these RHEB-deficient memory-like T cells failed to generate recall responses as the result of metabolic defects. While mTORC1 influenced CD8+ T cell effector responses, mTORC2 activity regulated CD8+ T cell memory. mTORC2 inhibition resulted in metabolic reprogramming, which enhanced the generation of CD8+ memory cells. Overall, these results define specific roles for mTORC1 and mTORC2 that link metabolism and CD8+ T cell effector and memory generation and suggest that these functions have the potential to be targeted for enhancing vaccine efficacy and antitumor immunity.

mTORC2 Deficiency in Myeloid Dendritic Cells Enhances Their Allogeneic Th1 and Th17 Stimulatory Ability after TLR4 Ligation In Vitro and In Vivo.

The mammalian/mechanistic target of rapamycin (mTOR) is a key integrative kinase that functions in two independent complexes, mTOR complex (mTORC) 1 and mTORC2. In contrast to the well-defined role of mTORC1 in dendritic cells (DC), little is known about the function of mTORC2. In this study, to our knowledge, we demonstrate for the first time an enhanced ability of mTORC2-deficient myeloid DC to stimulate and polarize allogeneic T cells. We show that activated bone marrow-derived DC from conditional Rictor(-/-) mice exhibit lower coinhibitory B7-H1 molecule expression independently of the stimulus and enhanced IL-6, TNF-α, IL-12p70, and IL-23 production following TLR4 ligation. Accordingly, TLR4-activated Rictor(-/-) DC display augmented allogeneic T cell stimulatory ability, expanding IFN-γ(+) and IL-17(+), but not IL-10(+) or CD4(+)Foxp3(+) regulatory T cells in vitro. A similar DC profile was obtained by stimulating Dectin-1 (C-type lectin family member) on Rictor(-/-) DC. Using novel CD11c-specific Rictor(-/-) mice, we confirm the alloreactive Th1 and Th17 cell-polarizing ability of endogenous mTORC2-deficient DC after TLR4 ligation in vivo. Furthermore, we demonstrate that proinflammatory cytokines produced by Rictor(-/-) DC after LPS stimulation are key in promoting Th1/Th17 responses. These data establish that mTORC2 activity restrains conventional DC proinflammatory capacity and their ability to polarize T cells following TLR and non-TLR stimulation. Our findings provide new insight into the role of mTORC2 in regulating DC function and may have implications for emerging therapeutic strategies that target mTOR in cancer, infectious diseases, and transplantation.

Cartilage-specific deletion of mTOR upregulates autophagy and protects mice from osteoarthritis.

OBJECTIVES: Mammalian target of rapamycin (mTOR) (a serine/threonine protein kinase) is a major repressor of autophagy, a cell survival mechanism. The specific in vivo mechanism of mTOR signalling in OA pathophysiology is not fully characterised. We determined the expression of mTOR and known autophagy genes in human OA cartilage as well as mouse and dog models of experimental OA. We created cartilage-specific mTOR knockout (KO) mice to determine the specific role of mTOR in OA pathophysiology and autophagy signalling in vivo. METHODS: Inducible cartilage-specific mTOR KO mice were generated and subjected to mouse model of OA. Human OA chondrocytes were treated with rapamycin and transfected with Unc-51-like kinase 1 (ULK1) siRNA to determine mTOR signalling. RESULTS: mTOR is overexpressed in human OA cartilage as well as mouse and dog experimental OA. Upregulation of mTOR expression co-relates with increased chondrocyte apoptosis and reduced expression of key autophagy genes during OA. Subsequently, we show for the first time that cartilage-specific ablation of mTOR results in increased autophagy signalling and a significant protection from destabilisation of medial meniscus (DMM)-induced OA associated with a significant reduction in the articular cartilage degradation, apoptosis and synovial fibrosis. Furthermore, we show that regulation of ULK1/adenosine monophosphate-activated protein kinase (AMPK) signalling pathway by mTOR may in part be responsible for regulating autophagy signalling and the balance between catabolic and anabolic factors in the articular cartilage. CONCLUSIONS: This study provides a direct evidence of the role of mTOR and its downstream modulation of autophagy in articular cartilage homeostasis.

The nuclear import of ribosomal proteins is regulated by mTOR.

Mechanistic target of rapamycin (mTOR) is a central component of the essential signaling pathway that regulates cell growth and proliferation by controlling anabolic processes in cells. mTOR exists in two distinct mTOR complexes known as mTORC1 and mTORC2 that reside mostly in cytoplasm. In our study, the biochemical characterization of mTOR led to discovery of its novel localization on nuclear envelope where it associates with a critical regulator of nuclear import Ran Binding Protein 2 (RanBP2). We show that association of mTOR with RanBP2 is dependent on the mTOR kinase activity that regulates the nuclear import of ribosomal proteins. The mTOR kinase inhibitors within thirty minutes caused a substantial decrease of ribosomal proteins in the nuclear but not cytoplasmic fraction. Detection of a nuclear accumulation of the GFP-tagged ribosomal protein rpL7a also indicated its dependence on the mTOR kinase activity. The nuclear abundance of ribosomal proteins was not affected by inhibition of mTOR Complex 1 (mTORC1) by rapamycin or deficiency of mTORC2, suggesting a distinctive role of the nuclear envelope mTOR complex in the nuclear import. Thus, we identified that mTOR in association with RanBP2 mediates the active nuclear import of ribosomal proteins.

Mammalian target of rapamycin complex 2 modulates αßTCR processing and surface expression during thymocyte development.

An efficient immune response relies on the presence of T cells expressing a functional TCR. Whereas the mechanisms generating TCR diversity for antigenic recognition are well defined, what controls its surface expression is less known. In this study, we found that deletion of the mammalian target of rapamycin complex (mTORC) 2 component rictor at early stages of T cell development led to aberrant maturation and increased proteasomal degradation of nascent TCRs. Although CD127 expression became elevated, the levels of TCRs as well as CD4, CD8, CD69, Notch, and CD147 were significantly attenuated on the surface of rictor-deficient thymocytes. Diminished expression of these receptors led to suboptimal signaling, partial CD4(-)CD8(-) double-negative 4 (CD25(-)CD44(-)) proliferation, and CD4(+)CD8(+) double-positive activation as well as developmental blocks at the CD4(-)CD8(-) double-negative 3 (CD25(+)CD44(-)) and CD8-immature CD8(+) single-positive stages. Because CD147 glycosylation was also defective in SIN1-deficient fibroblasts, our findings suggest that mTORC2 is involved in the co/posttranslational processing of membrane receptors. Thus, mTORC2 impacts development via regulation of the quantity and quality of receptors important for cell differentiation.

Mammalian target of rapamycin promotes oligodendrocyte differentiation, initiation and extent of CNS myelination.

Prior studies support a role for mammalian target of rapamycin (mTOR) signaling in oligodendrocyte differentiation and myelination. Here we use Cre-recombinase driven by the CNP promoter to generate a mouse line with oligodendrocyte-specific knockdown of mTOR (mTOR cKO) in the CNS. We provide evidence that mTOR is necessary for proper oligodendrocyte differentiation and myelination in the spinal cord. Specifically, the number of mature oligodendrocytes was reduced, and the initiation and extent of myelination were impaired during spinal cord development. Consistent with these data, myelin protein expression, including myelin basic protein, proteolipid protein, myelin oligodendrocyte glycoprotein, and myelin-associated glycoprotein, was delayed in the spinal cord. Hypomyelination of the spinal cord persisted into adulthood, as did the reduction in numbers of mature oligodendrocytes. In the cortex, the structure of myelin appeared normal during development and in the adult; however, myelin protein expression was delayed during development and was abnormal in the adult. Myelin basic protein was significantly reduced in all regions at postnatal day 25. These data demonstrate that mTOR promotes oligodendrocyte differentiation and CNS myelination in vivo and show that the requirement for mTOR varies by region with the spinal cord most dependent on mTOR.

Loss of mTOR complex 1 induces developmental blockage in early T-lymphopoiesis and eradicates T-cell acute lymphoblastic leukemia cells.

mTOR is an evolutionarily conserved kinase that plays a critical role in sensing and responding to environmental determinants. Recent studies have shown that fine-tuning of the activity of mTOR complexes contributes to organogenesis and tumorigenesis. Although rapamycin, an allosteric mTOR inhibitor, is an effective immunosuppressant, the precise roles of mTOR complexes in early T-cell development remain unclear. Here we show that mTORC1 plays a critical role in the development of both early T-cell progenitors and leukemia. Deletion of Raptor, an essential component of mTORC1, produced defects in the earliest development of T-cell progenitors in vivo and in vitro. Deficiency of Raptor resulted in cell cycle abnormalities in early T-cell progenitors that were associated with instability of the Cyclin D2/D3-CDK6 complexes; deficiency of Rictor, an mTORC2 component, did not have the same effect, indicating that mTORC1 and -2 control T-cell development in different ways. In a model of myeloproliferative neoplasm and T-cell acute lymphoblastic leukemia (T-ALL) evoked by Kras activation, Raptor deficiency dramatically inhibited the cell cycle in oncogenic Kras-expressing T-cell progenitors, but not myeloid progenitors, and specifically prevented the development of T-ALL. Although rapamycin treatment significantly prolonged the survival of recipient mice bearing T-ALL cells, rapamycin-insensitive leukemia cells continued to propagate in vivo. In contrast, Raptor deficiency in the T-ALL model resulted in cell cycle arrest and efficient eradication of leukemia. Thus, understanding the cell-context-dependent role of mTORC1 illustrates the potential importance of mTOR signals as therapeutic targets.

Rictor/mTORC2 protects against cisplatin-induced tubular cell death and acute kidney injury.

The mammalian target of rapamycin (mTOR) plays a critical role for cell growth and survival in many cell types. While substantial progress has been made in understanding the abnormal activation of mTORC1 in the pathogenesis of kidney disease, little is known about mTORC2 in kidney disease such as acute kidney injury (AKI). To study this, we generated a mouse model with tubule-specific deletion of Rictor (Tubule-Rictor-/-). The knockouts were born normal and no obvious kidney dysfunction or kidney morphologic abnormality was found within 2 months of birth. However, ablation of Rictor in the tubular cells exacerbated cisplatin-induced AKI compared to that in the control littermates. As expected, tubular cell apoptosis, Akt phosphorylation (Ser473), and autophagy were induced in the kidneys from the control littermates by cisplatin treatment. Less cell autophagy or Akt phosphorylation and more cell apoptosis in the kidneys of the knockout mice were identified compared with those in the control littermates. In NRK-52E cells in vitro, Rictor siRNA transfection sensitized cell apoptosis to cisplatin but with reduced cisplatin-induced autophagy. Metformin, an inducer of autophagy, abolished cell death induced by Rictor siRNA and cisplatin. Thus, endogenous Rictor/mTORC2 protects against cisplatin-induced AKI, probably mediated by promoting cell survival through Akt signaling activation and induction of autophagy.

Increased mammalian lifespan and a segmental and tissue-specific slowing of aging after genetic reduction of mTOR expression.

We analyzed aging parameters using a mechanistic target of rapamycin (mTOR) hypomorphic mouse model. Mice with two hypomorphic (mTOR(Δ/Δ)) alleles are viable but express mTOR at approximately 25% of wild-type levels. These animals demonstrate reduced mTORC1 and mTORC2 activity and exhibit an approximately 20% increase in median survival. While mTOR(Δ/Δ) mice are smaller than wild-type mice, these animals do not demonstrate any alterations in normalized food intake, glucose homeostasis, or metabolic rate. Consistent with their increased lifespan, mTOR(Δ/Δ) mice exhibited a reduction in a number of aging tissue biomarkers. Functional assessment suggested that, as mTOR(Δ/Δ) mice age, they exhibit a marked functional preservation in many, but not all, organ systems. Thus, in a mammalian model, while reducing mTOR expression markedly increases overall lifespan, it affects the age-dependent decline in tissue and organ function in a segmental fashion.

Akt is efficiently activated by PIF-pocket- and PtdIns(3,4,5)P3-dependent mechanisms leading to resistance to PDK1 inhibitors.

Mutations leading to inappropriate activation of Akt isoforms contribute to proliferation and survival of a significant proportion of human cancers. Akt is activated by phosphorylation of its T-loop residue (Thr(308)) by PDK1 (3-phosphoinositide-dependent kinase-1) and its C-terminal hydrophobic motif (Ser(473)) by mTORC2 [mTOR (mammalian target of rapamycin) complex 2]. Potent PDK1 inhibitors such as GSK2334470 have recently been elaborated as potential anti-cancer agents. However, these compounds were surprisingly ineffective at suppressing Akt activation. In the present study we demonstrate that resistance to PDK1 inhibitors results from Akt being efficiently recruited to PDK1 via two alternative mechanisms. The first involves ability of Akt and PDK1 to mutually interact with the PI3K (phosphoinositide 3-kinase) second messenger PtdIns(3,4,5)P3. The second entails recruitment of PDK1 to Akt after its phosphorylation at Ser(473) by mTORC2, via a substrate-docking motif termed the PIF-pocket. We find that disruption of either the PtdIns(3,4,5)P3 or the Ser(473) phosphorylation/PIF-pocket mechanism only moderately impacts on Akt activation, but induces marked sensitization to PDK1 inhibitors. These findings suggest that suppression of Ser(473) phosphorylation by using mTOR inhibitors would disrupt the PIF-pocket mechanism and thereby sensitize Akt to PDK1 inhibitors. Consistent with this, we find combing PDK1 and mTOR inhibitors reduced Akt activation to below basal levels and markedly inhibited proliferation of all of the cell lines tested. Our results suggest further work is warranted to explore the utility of combining PDK1 and mTOR inhibitors as a therapeutic strategy for treatment of cancers that harbour mutations elevating Akt activity.