Constitutive mTOR activation in TSC mutants sensitizes cells to energy starvation and genomic damage via p53

Miscoordination of growth and proliferation with the cellular stress response can lead to tumorigenesis. Mammalian target of rapamycin (mTOR), a central cell growth controller, is highly activated in some malignant neoplasms, and its clinical implications are under extensive investigation. We show that constitutive mTOR activity amplifies p53 activation, in vitro and in vivo, by stimulating p53 translation. Thus, loss of TSC1 or TSC2, the negative regulators of mTOR, results in dramatic accumulation of p53 and apoptosis in response to stress conditions. In other words, the inactivation of mTOR prevents cell death by nutrient stress and genomic damage via p53. Consistently, we also show that p53 is elevated in TSC tumors, which rarely become malignant. The coordinated relationship between mTOR and p53 during cellular stress provides a possible explanation for the benign nature of hamartoma syndromes, including TSC. Clinically, this also suggests that the efficacy of mTOR inhibitors in anti-neoplastic therapy may also depend on p53 status, and mTOR inhibitors may antagonize the effects of genotoxic chemotherapeutics.     

SIRT1 Negatively Regulates the Mammalian Target of Rapamycin

The IGF/mTOR pathway, which is modulated by nutrients, growth factors, energy status and cellular stress regulates aging in various organisms. SIRT1 is a NAD+ dependent deacetylase that is known to regulate caloric restriction mediated longevity in model organisms, and has also been linked to the insulin/IGF signaling pathway. Here we investigated the potential regulation of mTOR signaling by SIRT1 in response to nutrients and cellular stress. We demonstrate that SIRT1 deficiency results in elevated mTOR signaling, which is not abolished by stress conditions. The SIRT1 activator resveratrol reduces, whereas SIRT1 inhibitor nicotinamide enhances mTOR activity in a SIRT1 dependent manner. Furthermore, we demonstrate that SIRT1 interacts with TSC2, a component of the mTOR inhibitory-complex upstream to mTORC1, and regulates mTOR signaling in a TSC2 dependent manner. These results demonstrate that SIRT1 negatively regulates mTOR signaling potentially through the TSC1/2 complex.     

The TSC1 and TSC2 tumor suppressors are required for proper ER stress response and protect cells from ER stress-induced apoptosis

Tuberous sclerosis complex (TSC)1 and TSC2 are tumor suppressors that inhibit cell growth and mutation of either gene causes benign tumors in multiple tissues. The TSC1 and TSC2 gene products form a functional complex that has GTPase-activating protein (GAP) activity toward Ras homolog enriched in brain (Rheb) to inhibit mammalian target of rapamycin complex 1 (mTORC1), which is constitutively activated in TSC mutant tumors. We found that cells with mutation in either TSC1 or TSC2 are hypersensitive to endoplasmic reticulum (ER) stress and undergo apoptosis. Although the TSC mutant cells show elevated eIF2α phosphorylation, an early ER stress response marker, at both basal and induced conditions, induction of other ER stress response markers, including ATF4, ATF6 and C/EBP homologous protein (CHOP), is severely compromised. The defects in ER stress response are restored by raptor knockdown but not by rapamycin treatment in the TSC mutant cells, indicating that a rapamycin-insensitive mTORC function is responsible for the defects in ER stress response. Consistently, activation of Rheb sensitizes cells to ER stress. Our data show an important role of TSC1/TSC2 and Rheb in unfolded protein response and cell survival. We speculate that an important physiological function of the TSC1/2 tumor suppressors is to protect cells from harmful conditions. These observations indicate a potential therapeutic application of using ER stress agents to selectively kill TSC1 or TSC2 mutant cells for TSC treatment.     

Regulation of mTOR and cell growth in response to energy stress by REDD1

The tuberous sclerosis tumor suppressors TSC1 and TSC2 regulate the mTOR pathway to control translation and cell growth in response to nutrient and growth factor stimuli. We have recently identified the stress response REDD1 gene as a mediator of tuberous sclerosis complex (TSC)-dependent mTOR regulation by hypoxia. Here, we demonstrate that REDD1 inhibits mTOR function to control cell growth in response to energy stress. Endogenous REDD1 is induced following energy stress, and REDD1-/- cells are highly defective in dephosphorylation of the key mTOR substrates S6K and 4E-BP1 following either ATP depletion or direct activation of the AMP-activated protein kinase (AMPK). REDD1 likely acts on the TSC1/2 complex, as regulation of mTOR substrate phosphorylation by REDD1 requires TSC2 and is blocked by overexpression of the TSC1/2 downstream target Rheb but is not blocked by inhibition of AMPK. Tetracycline-inducible expression of REDD1 triggers rapid dephosphorylation of S6K and 4E-BP1 and significantly decreases cellular size. Conversely, inhibition of endogenous REDD1 by short interfering RNA increases cell size in a rapamycin-sensitive manner, and REDD1-/- cells are defective in cell growth regulation following ATP depletion. These results define REDD1 as a critical transducer of the cellular response to energy depletion through the TSC-mTOR pathway.     

Autophagy

Mammalian target of rapamycin (mTOR) complex 1 (mTORC1), which is activated in tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM), is a master regulator of cell growth, cellular metabolism, and autophagy. Treatment of TSC and LAM patients with mTORC1 inhibitors partially decreases the size of brain and kidney tumors, and stabilizes pulmonary function. However, the tumors regrow and lung function continues to decline when treatment is discontinued. We hypothesized that dysregulation of autophagy plays a critical role in the pathogenesis of tumors with mTORC1 hyperactivation and in their response to mTORC1-targeted therapy. We found that cells lacking TSC2 have low levels of autophagy under basal and cellular stress conditions. Using genetic and pharmacological approaches, we discovered that the survival of Tsc2-deficient tumor cells is dependent on autophagy induction. Thus, autophagy inhibitors may have therapeutic potential in TSC and LAM, either as single agent therapy or in combination with mTORC1 inhibitors.     

Autophagy: an 'Achilles' heel of tumorigenesis in TSC and LAM

Mammalian target of rapamycin (mTOR) complex 1 (mTORC1), which is activated in tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM), is a master regulator of cell growth, cellular metabolism, and autophagy. Treatment of TSC and LAM patients with mTORC1 inhibitors partially decreases the size of brain and kidney tumors, and stabilizes pulmonary function. However, the tumors regrow and lung function continues to decline when treatment is discontinued. We hypothesized that dysregulation of autophagy plays a critical role in the pathogenesis of tumors with mTORC1 hyperactivation and in their response to mTORC1-targeted therapy. We found that cells lacking TSC2 have low levels of autophagy under basal and cellular stress conditions. Using genetic and pharmacological approaches, we discovered that the survival of Tsc2-deficient tumor cells is dependent on autophagy induction. Thus, autophagy inhibitors may have therapeutic potential in TSC and LAM, either as single agent therapy or in combination with mTORC1 inhibitors.     

Phosphorylation of Raptor by p38beta participates in arsenite-induced mammalian target of rapamycin complex 1 (mTORC1) activation

Cell growth is influenced by environmental stress. Mammalian target of rapamycin (mTOR), the central regulator of cell growth, can be positively or negatively regulated by various stresses through different mechanisms. The p38 MAP kinase pathway is essential in cellular stress responses. Activation of MK2, a downstream kinase of p38α, enhances mTOR complex 1 (mTORC1) activity by preventing TSC2 from inhibiting mTOR activation. The p38β-PRAK cascade targets Rheb to inhibit mTORC1 activity upon glucose depletion. Here we show the activation of p38β participates in activation of mTOR complex 1 (mTORC1) induced by arsenite but not insulin, nutrients, anisomycin, or H(2)O(2). Arsenite treatment of cells activates p38β and induces interaction between p38β and Raptor, a regulatory component of mTORC1, resulting in phosphorylation of Raptor on Ser(863) and Ser(771). The phosphorylation of Raptor on these sites enhances mTORC1 activity, and contributes largely to arsenite-induced mTORC1 activation. Our results shown here and in previous work demonstrate that the p38 pathway can regulate different components of the mTORC1 pathway, and that p38β can target different substrates to either positively or negatively regulate mTORC1 activation when a cell encounters different environmental stresses.     

Inappropriate activation of the TSC/Rheb/mTOR/S6K cassette induces IRS1/2 depletion, insulin resistance, and cell survival deficiencies

Tuberous sclerosis is a largely benign tumor syndrome derived from the acquisition of somatic lesions in genes encoding the tumor suppressor products, TSC1 or TSC2. Loss of function of the TSC1-TSC2 complex, which acts as a Rheb GAP, yields constitutive, unrestrained signaling from the cell growth machinery comprised of Rheb, mTOR, and S6K. We demonstrate herein that constitutive activation of the Rheb/mTOR/S6K cassette, whether by genetic deletion of TSC1 or TSC2 or by ectopic expression of Rheb, is sufficient to induce insulin resistance. This is the result of downregulation of the insulin receptor substrates, IRS1 and IRS2, which become limiting for signal transmission from the insulin receptor to PI3K. Downstream of PI3K, the survival kinase, Akt, is completely refractory to activation by IRS-dependent growth factor pathways such as insulin or IGF-I in TSC1- or TSC2-deficient cells but not to activation by IRS-independent pathways such as those utilized by PDGF. The antiapoptotic program induced by IGF-I but not PDGF is severely compromised in TSC2 null cells. Our results suggest that inappropriate activation of the Rheb/mTOR/S6K pathway imposes a negative feedback program to attenuate IRS-dependent processes such as cell survival.     

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.     

The stress-inducted proteins RTP801 and RTP801L are negative regulators of the mammalian target of rapamycin pathway

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that plays an essential role in cell growth control. mTOR stimulates cell growth by phosphorylating p70 ribosomal S6 kinase (S6K) and eukaryote initiation factor 4E-binding protein 1 (4EBP1). The mTOR pathway is regulated by a wide variety of cellular signals, including mitogenic growth factors, nutrients, cellular energy levels, and stress conditions. Recent studies have proposed several mechanisms to explain how mTOR is regulated by growth factors and cellular energy levels. However, little is known as to how mTOR is regulated by stress conditions. We observed that two stress-induced proteins, RTP801/Redd1 and RTP801L/Redd2, potently inhibit signaling through mTOR. Our data support that RTP801 and RTP801L work downstream of AKT and upstream of TSC2 to inhibit mTOR functions. These results add a new dimension to mTOR pathway regulation and provide a possible molecular mechanism of how cellular stress conditions may regulate mTOR function.     

TSC2: filling the GAP in the mTOR signaling pathway

The tumor-suppressor proteins TSC1 and TSC2 are associated with an autosomal dominant disorder known as tuberous sclerosis complex (TSC). TSC1 and TSC2 function as a heterodimer to inhibit cell growth and proliferation. Another protein, mTOR (mammalian target of rapamycin), is regarded as a central controller of cell growth in response to growth factors, cellular energy and nutrient levels. Recent breakthroughs in TSC research link the TSC1/2 heterodimer protein to the mTOR signaling network. It has recently been shown that TSC2 has GTPase-activating protein (GAP) activity towards the Ras family small GTPase Rheb (Ras homolog enriched in brain), and TSC1/2 antagonizes the mTOR signaling pathway via stimulation of GTP hydrolysis of Rheb. Thus, TSC1/2 and Rheb have pivotal roles in mediating growth factors, nutrient and energy sensing signals to mTOR-dependent targets. These discoveries lend new insight into TSC pathogenesis.     

Differential involvement of IkappaB kinases alpha and beta in cytokine- and insulin-induced mammalian target of rapamycin activation determined by Akt

The mammalian target of rapamycin (mTOR) is a mediator of cell growth, survival, and energy metabolism at least partly through its ability to regulate mRNA translation. mTOR is activated downstream of growth factors such as insulin, cytokines such as TNF, and Akt-dependent signaling associated with oncoprotein expression. mTOR is negatively controlled by the tuberous sclerosis complex 1/2 (TSC1/2), and activation of Akt induces phosphorylation of TSC2, which blocks the repressive TSC1/2 activity. Previously, we showed that activation of mTOR in PTEN-deficient cancer cells involves IkappaB kinase (IKK) alpha, a catalytic subunit of the IKK complex that controls NF-kappaB activation. Recently, a distinct IKK subunit, IKKbeta, was shown to phosphorylate TSC1 to promote mTOR activation in an Akt-independent manner in certain cells stimulated with TNF and in some cancer cells. In this study, we have explored the involvement of both IKKalpha and IKKbeta in insulin- and TNF-induced mTOR activation. Insulin activation of mTOR requires Akt in a manner that involves IKKalpha, preferentially to IKKbeta, and TSC2 phosphorylation. TNF, in most cells examined, activates Akt to use IKKalpha to control mTOR activation. In MCF7 cells, TNF does not activate Akt and requires IKKbeta to activate mTOR. The results show that Akt-dependent signaling, induced by cytokines or insulin, alters the IKK subunit-dependent control of mTOR.     

Efficacy of combined inhibition of mTOR and ERK/MAPK pathways in treating a tuberous sclerosis complex cell model

Tuberous sclerosis complex （TSC） is an autosomal dominant tumor syndrome which afflicts multiple organs and for which there is no cure, such that TSC patients may develop severe mental retardation and succumb to renal or respiratory failure. TSC derives from inacti- vating mutations of either the TSC1 or TSC2 tumor suppressor gene, and the resulting inactivation of the TSC1/TSC2 protein complex causes hyperactivation of the mammalian target of rapamyein （mTOR）, leading to uncontrolled cell growth and proliferation. Recent clinical trials of targeted suppression of mTOR have yielded only modest success in TSC patients. It was proposed that abrogation of a newly identified mTOR-mediated negative feedback regulation on extracellular signal-regulated kinase/mitogen-activated protein kinase （ERK/MAPK） signaling pathway and on the well-documented RTK-PI3K-AKT signaling cascade could limit the efficacy of mTOR inhibitors in the treatment of TSC patients. Therefore, we speculate that dual inhibition of mTOR and ERK/MAPK pathways may overcome the disadvantage of single agent therapies and boost the efficacy of mTOR targeted therapies for TSC patients. Investigation of this hypothesis in a TSC cell model revealed that mTOR suppression with an mTOR inhibitor, rapamycin （sirolimus）, led to up-regulation of ERK/MAPK signaling in mouse Tsc2 knockout cells and that this augmented signaling was attenuated by concurrent administration of a MEK1/2 inhibitor, PD98059. When compared with monotherapy, combinatorial application of rapamycin and PD98059 had greater inhibitory effects on Tsc2 deficient cell proliferation, suggesting that combined suppression of mTOR and ERK/MAPK signaling pathways may have advantages over single mTOR inhibition in the treatment of TSC patients.     

The p53 target Plk2 interacts with TSC proteins impacting mTOR signaling,tumor growth,and chemosensitivity under hypoxic conditions

Tuberous sclerosis complex 1 (TSC1) inhibits mammalian target of rapamycin (mTOR), a central promotor of cell growth and proliferation. The protein product of the TSC1 gene, hamartin (referred to as TSC1) is known to interact with Polo-like kinase 1 (Plk1) in a cell cycle regulated, phosphorylation-dependent manner. We hypothesized that the p53 target gene, Plk2, is a tumor suppressor, mediating its tumor suppressor function through interactions with TSC1 that facilitate TSC1/2 restraint of mTOR under hypoxic stress. We found that human lung tumor cells deficient in Plk2 grew larger than control tumors, and that Plk2 interacts with endogenous TSC1 protein. Additionally, C-terminal Plk2-GST fusion protein bound both TSC1 and TSC2 proteins. TSC1 levels were elevated in response to Adriamycin and cells transiently over-expressing Plk2 demonstrated decreased phosphorylation of the downstream target of mTOR, ribosomal protein p70S6 kinase during hypoxia. Plk2 levels were inversely correlated with cytoplasmic p70S6K phosphorylation. Plk2 levels did not increase in response to DNA damage (Adriamycin, CPT-11) when HCT 116 and H460 cells were exposed to hypoxia. TSC1-deficient mouse embryonic fibroblasts with TSC1 added back demonstrated decreased S6K phosphorylation, which was further decreased when Plk2 was transiently over-expressed. Interestingly, under normoxia, Plk2 deficient tumor cells demonstrated increased apoptosis in response to various chemotherapeutic agents including CPT-11 but increased resistance to apoptotic death after CPT-11 treatment under hypoxia, and tumor xenografts comprised of these Plk2-deficient cells were resistant to CPT-11. Our results point to a novel Plk2-TSC1 interaction with effects on mTOR signaling during hypoxia, and tumor growth that may enable targeting Plk2 signaling in cancer therapy.     

The tuberous sclerosis protein TSC2 is not required for the regulation of the mammalian target of rapamycin by amino acids and certain cellular stresses

Amino acids positively regulate signaling through the mammalian target of rapamycin (mTOR). Recent work demonstrated the importance of the tuberous sclerosis protein TSC2 for regulation of mTOR by insulin. TSC2 contains a GTPase-activator domain that promotes hydrolysis of GTP bound to Rheb, which positively regulates mTOR signaling. Some studies have suggested that TSC2 also mediates the control of mTOR by amino acids. In cells lacking TSC2, amino acid withdrawal still results in dephosphorylation of S6K1, ribosomal protein S6, the eukaryotic initiation factor 4E-binding protein, and elongation factor-2 kinase. The effects of amino acid withdrawal are diminished by inhibiting protein synthesis or adding back amino acids. These studies demonstrate that amino acid signaling to mTOR occurs independently of TSC2 and involves additional unidentified inputs. Although TSC2 is not required for amino acid control of mTOR, amino acid withdrawal does decrease the proportion of Rheb in the active GTP-bound state. Here we also show that Rheb and mTOR form stable complexes, which are not, however, disrupted by amino acid withdrawal. Mutants of Rheb that cannot bind GTP or GDP can interact with mTOR complexes. We also show that the effects of hydrogen peroxide and sorbitol, cell stresses that impair mTOR signaling, are independent of TSC2. Finally, we show that the ability of energy depletion (which impairs mTOR signaling in TSC2+/+ cells) to increase the phosphorylation of eukaryotic elongation factor 2 is also independent of TSC2. This likely involves the phosphorylation of the elongation factor-2 kinase by the AMP-activated protein kinase.     

Muscle atrophy in transgenic mice expressing a human TSC1 transgene

Muscle mass is regulated by a wide range of hormonal and nutritional signals, such as insulin and IGF. Tuberous sclerosis complex (TSC) is an inherited hamartoma disease with tumor growth in numerous organs. TSC is caused by mutation in either TSC1 or TSC2 tumor suppressor genes that negatively regulate insulin-induced S6K activation and cell growth. Here we report that expression of human TSC1 (hTSC1) in mouse skeletal muscle leads to reduction of muscle mass. Expression of hTSC1 stabilizes endogenous TSC2 and leads to inhibition of the mTOR signaling. The hTSC1-mTSC2 hetero-complex and its downstream components remain sensitive to insulin stimulation and nutrition signals. This study suggests that an increase in the steady state level of resident TSC1-TSC2 complex is sufficient to reduce muscle mass and cause atrophy.     

TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling

Tuberous sclerosis (TSC) is an autosomal dominant disorder characterized by the formation of hamartomas in a wide range of human tissues. Mutation in either the TSC1 or TSC2 tumour suppressor gene is responsible for both the familial and sporadic forms of this disease. TSC1 and TSC2 proteins form a physical and functional complex in vivo. Here, we show that TSC1-TSC2 inhibits the p70 ribosomal protein S6 kinase 1 (an activator of translation) and activates the eukaryotic initiation factor 4E binding protein 1 (4E-BP1, an inhibitor of translational initiation). These functions of TSC1-TSC2 are mediated by inhibition of the mammalian target of rapamycin (mTOR). Furthermore, TSC2 is directly phosphorylated by Akt, which is involved in stimulating cell growth and is activated by growth stimulating signals, such as insulin. TSC2 is inactivated by Akt-dependent phosphorylation, which destabilizes TSC2 and disrupts its interaction with TSC1. Our data indicate a molecular mechanism for TSC2 in insulin signalling, tumour suppressor functions and in the inhibition of cell growth.