Akt-dependent and -independent mechanisms of mTOR regulation in cancer

The protein kinase mTOR (mammalian target of rapamycin) is a critical regulator of cellular metabolism, growth, and proliferation. These processes contribute to tumor formation, and many cancers are characterized by aberrant activation of mTOR. Although activating mutations in mTOR itself have not been identified, deregulation of upstream components that regulate mTOR are prevalent in cancer. The prototypic mechanism of mTOR regulation in cells is through activation of the PI3K/Akt pathway, but mTOR receives input from multiple signaling pathways. This review will discuss Akt-dependent and -independent mechanisms of mTOR regulation in response to mitogenic signals, as well as its regulation in response to energy and nutrient-sensing pathways. Preclinical and clinical studies have demonstrated that tumors bearing genetic alterations that activate mTOR are sensitive to pharmacologic inhibition of mTOR. Elucidation of novel pathways that regulate mTOR may help identify predictive factors for sensitivity to mTOR inhibitors, and could provide new therapeutic targets for inhibiting the mTOR pathway in cancer. This review will also highlight pharmacologic approaches that inhibit mTOR via activation of the AMP-activated protein kinase (AMPK), an important inhibitor of the mTOR pathway and an emerging target in cancer.     

mTOR Inhibition Induces Compensatory,Therapeutically Targetable MEK Activation in Renal Cell Carcinoma

Rapamycin derivatives allosterically targeting mTOR are currently FDA approved to treat advanced renal cell carcinoma (RCC), and catalytic inhibitors of mTOR/PI3K are now in clinical trials for treating various solid tumors. We sought to investigate the relative efficacy of allosteric versus catalytic mTOR inhibition, evaluate the crosstalk between the mTOR and MEK/ERK pathways, as well as the therapeutic potential of dual mTOR and MEK inhibition in RCC. Pharmacologic (rapamycin and BEZ235) and genetic manipulation of the mTOR pathway were evaluated by in vitro assays as monotherapy as well as in combination with MEK inhibition (GSK1120212). Catalytic mTOR inhibition with BEZ235 decreased proliferation and increased apoptosis better than allosteric mTOR inhibition with rapamycin. While mTOR inhibition upregulated MEK/ERK signaling, concurrent inhibition of both pathways had enhanced therapeutic efficacy. Finally, primary RCC tumors could be classified into subgroups [(I) MEK activated, (II) Dual MEK and mTOR activated, (III) Not activated, and (IV) mTOR activated] based on their relative activation of the PI3K/mTOR and MEK pathways. Patients with mTOR only activated tumors had the worst prognosis. In summary, dual targeting of the mTOR and MEK pathways in RCC can enhance therapeutic efficacy and primary RCC can be subclassified based on their relative levels of mTOR and MEK activation with potential therapeutic implications.     

GbetaL,a positive regulator of the rapamycin-sensitive pathway required for the nutrient-sensitive interaction between raptor and mTOR

mTOR and raptor are components of a signaling pathway that regulates mammalian cell growth in response to nutrients and growth factors. Here, we identify a member of this pathway, a protein named GbetaL that binds to the kinase domain of mTOR and stabilizes the interaction of raptor with mTOR. Like mTOR and raptor, GbetaL participates in nutrient- and growth factor-mediated signaling to S6K1, a downstream effector of mTOR, and in the control of cell size. The binding of GbetaL to mTOR strongly stimulates the kinase activity of mTOR toward S6K1 and 4E-BP1, an effect reversed by the stable interaction of raptor with mTOR. Interestingly, nutrients and rapamycin regulate the association between mTOR and raptor only in complexes that also contain GbetaL. Thus, we propose that the opposing effects on mTOR activity of the GbetaL- and raptor-mediated interactions regulate the mTOR pathway.     

The functions of mTOR in ischemic diseases

Mammalian Target of Rapamycin (mTOR) is a serine/threonine kinase and that forms two multiprotein complexes known as the mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTOR regulates cell growth, proliferation and survival. mTORC1 is composed of the mTOR catalytic subunit and three associated proteins: raptor, mLST8/GβL and PRAS40. mTORC2 contains mTOR, rictor, mLST8/GβL, mSin1, and protor. Here, we discuss mTOR as a promising anti-ischemic agent. It is believed that mTORC2 lies down-stream of Akt and acts as a direct activator of Akt. The different functions of mTOR can be explained by the existence of two distinct mTOR complexes containing unique interacting proteins. The loss of TSC2, which is upstream of mTOR, activates S6K1, promotes cell growth and survival, activates mTOR kinase activities, inhibits mTORC1 and mTORC2 via mTOR inhibitors, and suppresses S6K1 and Akt. Although mTOR signaling pathways are often activated in human diseases, such as cancer, mTOR signaling pathways are deactivated in ischemic diseases. From Drosophila to humans, mTOR is necessary for Ser473 phosphorylation of Akt, and the regulation of Akt-mTOR signaling pathways may have a potential role in ischemic disease. This review evaluates the potential functions of mTOR in ischemic diseases. A novel mTOR-interacting protein deregulates over-expression in ischemic disease, representing a new mechanism for controlling mTOR signaling pathways and potential therapeutic strategies for ischemic diseases.     

Mechanistic target of rapamycin controls homeostasis of adipogenesis

Signaling mediated by the mechanistic target of rapamycin (mTOR) is believed to play a critical and positive role in adipogenesis, based on pharmacological evidence and genetic manipulation of mTOR regulators and targets. However, there is no direct genetic evidence for an autonomous role of mTOR itself in preadipocyte differentiation. To seek such evidence, we employed a conditional knockdown approach to deplete mTOR in preadipocytes. Surprisingly, while knockdown of S6K1, a target of mTOR, impairs 3T3-L1 preadipocyte differentiation, reduction of mTOR levels leads to increased differentiation. This enhanced adipogenesis requires the remaining mTOR activity, as mTOR inhibitors abolish differentiation in the mTOR knockdown cells. We also found that mTOR knockdown elevates the levels of CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ). Furthermore, partial reduction of mTOR levels alleviates inhibition of Akt by mTORC1 via IRS1, while at the same time maintaining its positive input through mTORC1 into the adipogenic program. The greater sensitivity of the IRS1-Akt pathway to mTOR levels provides a mechanism that explains the net outcome of enhanced adipogenesis through PPARγ upon mTOR knockdown. Our observations reveal an unexpected role of mTOR in suppressing adipogenesis and suggest that mTOR governs the homeostasis of the adipogenic process by modulating multiple signaling pathways.     

The expanding relevance of nuclear mTOR in carcinogenesis

Deregulated mTOR signaling drives the growth of various human cancers, making mTOR a major target for development of cancer chemotherapeutics. The role of mTOR in carcinogenesis is thought to be largely a consequence of its activity in the cytoplasm resulting in increased translation of pro-tumorigenic genes. However, emerging data locate mTOR in various subcellular compartments including Golgi, mitochondria, endoplasmic reticulum, and the nucleus, implying the presence of compartment-specific mTOR substrates and functions. Efforts to identify mTOR substrates in these compartments, and the mechanisms by which mTOR recruits these substrates and affects downstream cellular processes, will add to our understanding of the diversity of roles played by mTOR in carcinogenesis.     

mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery

mTOR/RAFT1/FRAP is the target of the immunosuppressive drug rapamycin and the central component of a nutrient- and hormone-sensitive signaling pathway that regulates cell growth. We report that mTOR forms a stoichiometric complex with raptor, an evolutionarily conserved protein with at least two roles in the mTOR pathway. Raptor has a positive role in nutrient-stimulated signaling to the downstream effector S6K1, maintenance of cell size, and mTOR protein expression. The association of raptor with mTOR also negatively regulates the mTOR kinase activity. Conditions that repress the pathway, such as nutrient deprivation and mitochondrial uncoupling, stabilize the mTOR-raptor association and inhibit mTOR kinase activity. We propose that raptor is a missing component of the mTOR pathway that through its association with mTOR regulates cell size in response to nutrient levels.     

mTOR phosphorylated at S2448 binds to raptor and rictor

In mammalian cells, the mammalian target of rapamycin (mTOR) forms an enzyme complex with raptor (together with other proteins) named mTOR complex 1 (mTORC1), of which a major target is the p70 ribosomal protein S6 kinase (p70S6K). A second enzyme complex, mTOR complex 2 (mTORC2), contains mTOR and rictor and regulates the Akt kinase. Both mTORC1 and mTORC2 are regulated by phosphorylation, complex formation and localization. So far, the role of p70S6K-mediated mTOR S2448 phosphorylation has not been investigated in detail. Here, we report that endogenous mTOR phosphorylated at S2448 binds to both, raptor and rictor. Experiments with chemical inhibitors of the mTOR kinase and of the phosphatidylinositol-3-kinase revealed that downregulation of mTOR S2448 phosphorylation correlates with decreased mTORC1 activity but can occur decoupled of effects on mTORC2 activity. In addition, we found that the correlation of the mTOR S2448 phosphorylation status with mTORC1 activity is not a consequence of effects on the assembly of mTOR protein and raptor. Our data allow new insights into the role of mTOR phosphorylation for the regulation of its kinase activity.     

mTOR signaling for biological control and cancer

Mammalian target of rapamycin (mTOR) is a major intersection that connects signals from the extracellular milieu to corresponding changes in intracellular processes. When abnormally regulated, the mTOR signaling pathway is implicated in a wide spectrum of cancers, neurological diseases, and proliferative disorders. Therefore, pharmacological agents that restore the regulatory balance of the mTOR pathway could be beneficial for a great number of diseases. This review summarizes current understanding of mTOR signaling and some unanswered questions in the field. We describe the composition of the mTOR complexes, upstream signals that activate mTOR, and physiological processes that mTOR regulates. We also discuss the role of mTOR and its downstream effectors in cancer, obesity and diabetes, and autism. J. Cell. Physiol. 228: 1658–1664, 2013. © 2013 Wiley Periodicals, Inc.     

Targeting the mTOR signaling network in cancer

The mammalian target of rapamycin (mTOR) is an unconventional protein kinase that is centrally involved in the control of cancer cell metabolism, growth and proliferation. The mTOR pathway has attracted broad scientific and clinical interest, particularly in light of the ongoing clinical cancer trials with mTOR inhibitors. The mixed clinical results to date reflect the complexity of both cancer as a disease target, and the mTOR signaling network, which contains two functionally distinct mTOR complexes, parallel regulatory pathways, and feedback loops that contribute to the variable cellular responses to the current inhibitors. In this review, we discuss the regulatory pathways that govern mTOR activity, and highlight clinical results obtained with the first generation of mTOR inhibitors to reach the oncology clinics.     

PLD2 forms a functional complex with mTOR/raptor to transduce mitogenic signals

Mammalian target-of-rapamycin (mTOR), which is a master controller of cell growth, senses a mitogenic signal in part through the lipid second messenger phosphatidic acid (PA), generated by phospholipase D (PLD). To understand further which isozymes of PLD are involved in this process, we compared the effect of PLD isozymes on mTOR activation. We found that PLD2 has an essential role in mitogen-induced mTOR activation as the siRNA-mediated knockdown of PLD2, not of PLD1, profoundly reduced the phosphorylations of S6K1 and 4EBP1, well-known mTOR effectors. Furthermore, exogenous PA-induced mTOR activation was abrogated by PLD2 knockdown, but not by PLD1 knockdown. This abrogation was found to be the result of complex formation between PLD2 and mTOR/raptor. PLD2 possesses a TOS-like motif (Phe-Glu-Val-Gln-Val, a.a. 265–269), through which it interacts with raptor independently of the other TOS motif-containing proteins, S6K1 and 4EBP1. PLD2-dependent mTOR activation appears to require PLD2 binding to mTOR/raptor with lipase activity, since lipase-inactive PLD2 cannot trigger mTOR activation despite its ability to interact with mTOR/raptor. Abrogation of mitogen-dependent mTOR activation by PLD2 knockdown was rescued only by wild type PLD2, but not by raptor binding-deficient and lipase-inactive PLD2. Our results demonstrate the importance of localized PA generation for the mitogen-induced activation of mTOR, which is achieved by a specific interaction between PLD2 and mTOR/raptor.     

Naturally secreted amyloid-beta increases mammalian target of rapamycin (mTOR) activity via a PRAS40-mediated mechanism

Reducing the mammalian target of rapamycin (mTOR) activity increases lifespan and health span in a variety of organisms. Alterations in protein homeostasis and mTOR activity and signaling have been reported in several neurodegenerative disorders, including Alzheimer disease (AD); however, the causes of such deregulations remain elusive. Here, we show that mTOR activity and signaling are increased in cell lines stably transfected with mutant amyloid precursor protein (APP) and in brains of 3xTg-AD mice, an animal model of AD. In addition, we show that in the 3xTg-AD mice, mTOR activity can be reduced to wild type levels by genetically preventing Aβ accumulation. Similarly, intrahippocampal injections of an anti-Aβ antibody reduced Aβ levels and normalized mTOR activity, indicating that high Aβ levels are necessary for mTOR hyperactivity in 3xTg-AD mice. We also show that the intrahippocampal injection of naturally secreted Aβ is sufficient to increase mTOR signaling in the brains of wild type mice. The mechanism behind the Aβ-induced mTOR hyperactivity is mediated by the proline-rich Akt substrate 40 (PRAS40) as we show that the activation of PRAS40 plays a key role in the Aβ-induced mTOR hyperactivity. Taken together, our data show that Aβ accumulation, which has been suggested to be the culprit of AD pathogenesis, causes mTOR hyperactivity by regulating PRAS40 phosphorylation. These data further indicate that the mTOR pathway is one of the pathways by which Aβ exerts its toxicity and further support the idea that reducing mTOR signaling in AD may be a valid therapeutic approach.     

Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40

Insulin stimulates protein synthesis and cell growth by activation of the protein kinases Akt (also known as protein kinase B, PKB) and mammalian target of rapamycin (mTOR). It was reported that Akt activates mTOR by phosphorylation and inhibition of tuberous sclerosis complex 2 (TSC2). However, in recent studies the physiological requirement of Akt phosphorylation of TSC2 for mTOR activation has been questioned. Here, we identify PRAS40 (proline-rich Akt/PKB substrate 40 kDa) as a novel mTOR binding partner that mediates Akt signals to mTOR. PRAS40 binds the mTOR kinase domain and its interaction with mTOR is induced under conditions that inhibit mTOR signalling, such as nutrient or serum deprivation or mitochondrial metabolic inhibition. Binding of PRAS40 inhibits mTOR activity and suppresses constitutive activation of mTOR in cells lacking TSC2. PRAS40 silencing inactivates insulin-receptor substrate-1 (IRS-1) and Akt, and uncouples the response of mTOR to Akt signals. Furthermore, PRAS40 phosphorylation by Akt and association with 14-3-3, a cytosolic anchor protein, are crucial for insulin to stimulate mTOR. These findings identify PRAS40 as an important regulator of insulin sensitivity of the Akt-mTOR pathway and a potential target for the treatment of cancers, insulin resistance and hamartoma syndromes.     

A Highlights from MBoC Selection: A Phosphatidylinositol 3-Kinase/Protein Kinase B-independent Activation of Mammalian Target of Rapamycin Signaling Is Sufficient to Induce Skeletal Muscle Hypertrophy

It has been widely proposed that signaling by mammalian target of rapamycin (mTOR) is both necessary and sufficient for the induction of skeletal muscle hypertrophy. Evidence for this hypothesis is largely based on studies that used stimuli that activate mTOR via a phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB)-dependent mechanism. However, the stimulation of signaling by PI3K/PKB also can activate several mTOR-independent growth-promoting events; thus, it is not clear whether signaling by mTOR is permissive, or sufficient, for the induction of hypertrophy. Furthermore, the presumed role of mTOR in hypertrophy is derived from studies that used rapamycin to inhibit mTOR; yet, there is very little direct evidence that mTOR is the rapamycin-sensitive element that confers the hypertrophic response. In this study, we determined that, in skeletal muscle, overexpression of Rheb stimulates a PI3K/PKB-independent activation of mTOR signaling, and this is sufficient for the induction of a rapamycin-sensitive hypertrophic response. Transgenic mice with muscle specific expression of various mTOR mutants also were used to demonstrate that mTOR is the rapamycin-sensitive element that conferred the hypertrophic response and that the kinase activity of mTOR is necessary for this event. Combined, these results provide direct genetic evidence that a PI3K/PKB-independent activation of mTOR signaling is sufficient to induce hypertrophy. In summary, overexpression of Rheb activates mTOR signaling via a PI3K/PKB-independent mechanism and is sufficient to induce skeletal muscle hypertrophy. The hypertrophic effects of Rheb are driven through a rapamycin-sensitive (RS) mechanism, mTOR is the RS element that confers the hypertrophy, and the kinase activity of mTOR is necessary for this event.     

Farnesylthiosalicylic acid inhibits mammalian target of rapamycin (mTOR) activity both in cells and in vitro by promoting dissociation of the mTOR-raptor complex

The mammalian target of rapamycin (mTOR) functions with raptor and mLST8 in a signaling complex that controls rates of cell growth and proliferation. Recent results indicate that an inhibitor of the Ras signaling pathway, farnesylthiosalicylic acid (FTS), decreased phosphorylation of the mTOR effectors, PHAS-I and S6K1, in breast cancer cells. Here we show that incubating 293T cells with FTS produced a stable change in mTOR activity that could be measured in immune complex kinase assays using purified PHAS-I as substrate. Similarly, FTS decreased the PHAS-I kinase activity of mTOR when added to cell extracts or to immune complexes containing mTOR. Incubating either cells or extracts with FTS also decreased the amount of raptor that coimmunoprecipitated with mTOR, although having relatively little effect on the amount of mLST8 that coimmunoprecipitated. The concentration effect curves of FTS for inhibition of mTOR activity and for dissociation of the raptor-mTOR complex were almost identical. Caffeine, wortmannin, LY294002, and rapamycin-FKBP12 also markedly inhibited mTOR activity in vitro, but unlike FTS, none of the other mTOR inhibitors appreciably changed the amount of raptor associated with mTOR. Thus, our findings indicate that FTS represents a new type of mTOR inhibitor, which acts by dissociating the functional mTOR-raptor signaling complex.     

HLA class I antibody-mediated endothelial cell proliferation via the mTOR pathway

Anti-HLA Abs have been shown to contribute to the process of transplant vasculopathy by binding to HLA class I molecules expressed by the endothelial and smooth muscle cells of the graft and transducing intracellular signals that elicit cell proliferation. The aim of this study was to determine the role of mammalian target of rapamycin (mTOR) in HLA class I-induced endothelial cell proliferation and to explore in depth the relationship between mTOR complexes and their downstream targets following ligation of HLA class I molecules by anti-HLA Abs. We used small interfering RNA technology to abrogate mTOR, rapamycin-insensitive companion of mTOR (rictor), or regulatory associated protein of mTOR (raptor) to study the function of these gene products to activate proteins involved in MHC class I-induced cell proliferation and survival. Knockdown of mTOR inhibited class I-mediated phosphorylation of proteins downstream of mTOR complex 1 and mTOR complex 2. Furthermore, knockdown of mTOR, rictor, or raptor blocked HLA class I-induced endothelial cell proliferation. Long-term pretreatment with the mTOR inhibitor rapamycin significantly blocked both mTOR-raptor and mTOR-rictor complex formation. Interestingly, rapamycin also blocked class I-induced Akt phosphorylation at Ser(473) and Bcl-2 expression. These results support the role of anti-HLA Abs in the process of transplant vasculopathy and suggest that exposure of the graft endothelium to anti-HLA Abs may promote proliferation through the mTOR pathway.     

Disruption of the mouse mTOR gene leads to early postimplantation lethality and prohibits embryonic stem cell development

The mammalian target of rapamycin (mTOR) is a key component of a signaling pathway which integrates inputs from nutrients and growth factors to regulate cell growth. Recent studies demonstrated that mice harboring an ethylnitrosourea-induced mutation in the gene encoding mTOR die at embryonic day 12.5 (E12.5). However, others have shown that the treatment of E4.5 blastocysts with rapamycin blocks trophoblast outgrowth, suggesting that the absence of mTOR should lead to embryonic lethality at an earlier stage. To resolve this discrepancy, we set out to disrupt the mTOR gene and analyze the outcome in both heterozygous and homozygous settings. Heterozygous mTOR (mTOR(+/-)) mice do not display any overt phenotype, although mouse embryonic fibroblasts derived from these mice show a 50% reduction in mTOR protein levels and phosphorylation of S6 kinase 1 T389, a site whose phosphorylation is directly mediated by mTOR. However, S6 phosphorylation, raptor levels, cell size, and cell cycle transit times are not diminished in these cells. In contrast to the situation in mTOR(+/-) mice, embryonic development of homozygous mTOR(-/-) mice appears to be arrested at E5.5; such embryos are severely runted and display an aberrant developmental phenotype. The ability of these embryos to implant corresponds to a limited level of trophoblast outgrowth in vitro, reflecting a maternal mRNA contribution, which has been shown to persist during preimplantation development. Moreover, mTOR(-/-) embryos display a lesion in inner cell mass proliferation, consistent with the inability to establish embryonic stem cells from mTOR(-/-) embryos.