TSC2-deficient Cells Research Articles

FoxOs Inhibit mTORC1 and Activate Akt by Inducing the Expression of Sestrin3 and Rictor

FoxO transcription factors and TORC1 are conserved downstream effectors of Akt. Here, we unraveled regulatory circuits underlying the interplay between Akt, FoxO, and mTOR. Activated FoxO1 inhibits mTORC1 by TSC2-dependent and TSC2-independent mechanisms. First, FoxO1 induces Sestrin3 (Sesn3) gene expression. Sesn3, in turn, inhibits mTORC1 activity in Tsc2-proficient cells. Second, FoxO1 elevates the expression of Rictor, leading to increased mTORC2 activity that consequently activates Akt. In Tsc2-deficient cells, the elevation of Rictor by FoxO increases mTORC2 assembly and activity at the expense of mTORC1, thereby activating Akt while inhibiting mTORC1. FoxO may act as a rheostat that maintains homeostatic balance between Akt and mTOR complexes' activities. In response to physiological stresses, FoxO maintains high Akt activity and low mTORC1 activity. Thus, under stress conditions, FoxO inhibits the anabolic activity of mTORC1, a major consumer of cellular energy, while activating Akt, which increases cellular energy metabolism, thereby maintaining cellular energy homeostasis.

TACC3-TSC2 maintains nuclear envelope structure and controls cell division

Studies of the role of tuberous sclerosis complex (TSC) proteins (TSC1/TSC2) in pathology have focused mainly on their capacity to regulate translation and cell growth, but their relationship with alterations of cellular structures and the cell cycle is not yet fully understood. The transforming acidic coiled-coil (TACC) domain-containing proteins are central players in structures and processes connected to the centrosome. Here, TACC3 interactome mapping identified TSC2 and 15 other physical interactors, including the evolutionary conserved interactions with ch-TOG/CKAP5 and FAM161B. TACC3 and TSC2 co-localize and co-purify with components of the nuclear envelope, and their deficiency causes morphological alterations of this structure. During cell division, TACC3 is necessary for the proper localization of phospho-Ser939 TSC2 at spindle poles and cytokinetic bridges. Accordingly, abscission alterations and increased frequency of binucleated cells were observed in Tacc3- and Tsc2-deficient cells relative to controls. In regulating cell division, TSC2 acts epistatically to TACC3 and, in addition to canonical TSC/mTOR signaling and cytokinetic associations, converges to the early mitotic checkpoint mediated by CHFR, consistently with nuclear envelope associations. Our findings link TACC3 to novel structural and cell division functions of TSC2, which may provide additional explanations for the clinical and pathological manifestations of lymphangioleiomyomatosis (LAM) disease and TSC syndrome, including the greater clinical severity of TSC2 mutations compared to TSC1 mutations.

MTOR Activation, Lymphangiogenesis, and Estrogen-Mediated Cell Survival: The “Perfect Storm” of Pro-Metastatic Factors in LAM Pathogenesis

Research interest in lymphangioleiomyomatosis (LAM) has grown dramatically in the past decade, particularly among cancer biologists. There are at least two reasons for this: first, the discovery in the year 2000 that LAM cells carry TSC2 gene mutations, linking LAM with cellular pathways including the PI3K/Akt/mTOR axis, and allowing the Tuberous Sclerosis Complex (TSC)-regulated pathways that are believed to underlie LAM pathogenesis to be studied in cells, yeast, Drosophila, and mice. A second reason for the rising interest in LAM is the discovery that LAM cells can travel to the lung, including repopulating a donor lung after lung transplantation, despite the fact that LAM cells are histologically benign. This "benign metastasis" underpinning suggests that elucidating LAM pathogenesis will unlock a set of fundamental mechanisms that underlie metastatic potential in the context of a cell that has not yet undergone malignant transformation. Here, we will outline the data supporting the metastatic model of LAM, consider the biochemical and cellular mechanisms that may enable LAM cells to metastasize, including both cell autonomous and non-cell autonomous factors, and highlight a mouse model in which estrogen promotes the metastasis and survival of TSC2-deficient cells in a MEK-dependent manner. We propose a multistep model of LAM cell metastasis that highlights multiple opportunities for therapeutic intervention. Taken together, the metastatic behavior of LAM cells and the involvement of tumor-related signaling pathways lead to optimism that cancer-related paradigms for diagnosis, staging, and therapy will lead to therapeutic breakthroughs for women living with LAM.

ER stress negatively regulates AKT/TSC/mTOR pathway to enhance autophagy

Disturbance to endoplasmic reticulum (ER) homeostasis that can not be rescued by the unfolded protein response (UPR) results in autophagy and cell death, but the precise mechanism was largely unknown. Here we demonstrated that ER stress-induced cell death was mediated by autophagy which was partly attributed to the inactivation of the mammalian target of rapamycin (mTOR). Three widely used ER stress inducers including tunicamycin, DTT and MG132 led to the conversion of LC3-I to LC3-II, a commonly used marker of autophagy, as well as the downregulation of mTOR concurrently. TSC-deficient cells with constitutive activation of mTOR exhibited more resistance to ER stress-induced autophagy, compared with their wild-type counterparts. Furthermore, our studies showed that ER stress-induced deactivation of mTOR was attributed to the downregulation of AKT/TSC/mTOR pathway. Phosphatase and tensin homolog (PTEN) and AMP-activated protein kinase (AMPK) as two regulators in this pathway seemed to be absent in this regulation. As a chemical chaperone helping the correct folding of proteins, 4-phenylbutyric acid (4-PBA) partly rescued AKT/TSC/mTOR pathway in drug-induced acute ER stress. Moreover, constitutively-activated mTOR-induced long-term ER stress attenuated RTK/PI3K/AKT signaling pathway in response to the stimulation by various growth factors, which could also be partly restored by 4-PBA.

MTORC1 Hyperactivity Inhibits Serum Deprivation-Induced Apoptosis via Increased Hexokinase II and GLUT1 Expression, Sustained Mcl-1 Expression, and Glycogen Synthase Kinase 3β Inhibition

The current concept is that Tsc-deficient cells are sensitized to apoptosis due to the inhibition of Akt activity by the negative feedback mechanism induced by the hyperactive mTORC1. Unexpectedly, however, we found that Tsc1/2-deficient cells exhibit increased resistance to serum deprivation-induced apoptosis. mTORC1 hyperactivity contributes to the apoptotic resistance of serum-deprived Tsc1/2-deficient cells in part by increasing the growth factor-independent expression of hexokinase II (HKII) and GLUT1. mTORC1-mediated increase in hypoxia-inducible factor 1alpha (HIF1alpha) abundance, which occurs in the absence of serum in normoxic Tsc2-deficient cells, contributes to these changes. Increased HIF1alpha abundance in these cells is attributed to both an increased level and the sustained translation of HIF1alpha mRNA. Sustained glycogen synthase kinase 3beta inhibition and Mcl-1 expression also contribute to the apoptotic resistance of Tsc2-deficient cells to serum deprivation. The inhibition of mTORC1 activity by either rapamycin or Raptor knockdown cannot resensitize these cells to serum deprivation-induced apoptosis because of elevated Akt activity that is an indirect consequence of mTORC1 inhibition. However, the increased HIF1alpha abundance and the maintenance of Mcl-1 protein expression in serum-deprived Tsc2(-/)(-) cells are dependent largely on the hyperactive eIF4E in these cells. Consistently, the reduction of eIF4E levels abrogates the resistance of Tsc2(-/)(-) cells to serum deprivation-induced apoptosis.

Signal Transducer and Activator of Transcription 3 Is Required for Abnormal Proliferation and Survival of TSC2-Deficient Cells: Relevance to Pulmonary Lymphangioleiomyomatosis

Tumor suppressor complex TSC1/TSC2 represents a key negative regulator of mammalian target of rapamycin (mTOR)-S6 kinase 1 signaling. Mutational inactivation of TSC1 or TSC2, linked to a rare lung disease, lymphangioleiomyomatosis (LAM), manifests as neoplastic growth of smooth-muscle (SM)-like cells and cystic destruction of the lungs that induces loss of pulmonary function. However, the precise mechanisms of abnormal cell growth in LAM remain uncertain. Here, we demonstrate increased signal transducer and activator of transcription (STAT) 3 expression, phosphorylation, and nuclear localization in SM-like cells in LAM lungs and in TSC2-null xenographic tumors. Treatment of TSC2-null tumors with mTOR inhibitor rapamycin attenuated STAT3 expression and phosphorylation. Increased STAT3 level and activation were also observed in LAM-dissociated (LAMD) cell cultures compared with normal human bronchus fibroblasts (HBFs) from LAM patients. Although interferon (IFN)-gamma inhibited proliferation of HBFs, IFN-gamma treatment had little effect on proliferation of LAMD and TSC2-null cells. Re-expression of TSC2 or treatment with rapamycin inhibited IFN-gamma-induced STAT3 phosphorylation and synergized with IFN-gamma in inhibiting TSC2-null and LAMD cell proliferation. Reduction of STAT3 protein levels or activity using specific small interfering RNA or inhibitory peptide, respectively, decreased proliferation and induced apoptosis in TSC2-null and LAMD cells and sensitized cells to growth-inhibitory and proapoptotic effects of IFN-gamma. Collectively, our data demonstrate that STAT3 activation is required for proliferation and survival of cells with TSC2 dysfunction, that STAT3 impedes growth-inhibitory and proapoptotic effects of IFN-gamma, and that TSC2- and rapamycin-dependent inhibition of STAT3 restores antiproliferative effects of IFN-gamma. Thus, STAT3 may provide a novel therapeutic target for diseases associated with TSC1/TSC2 dysfunction.

Rheb controls misfolded protein metabolism by inhibiting aggresome formation and autophagy

Perinuclear aggresome formation is a key mechanism to dispose of misfolded proteins that exceed the degradative capacity of ubiquitin-proteasome and autophagy-lysosome systems. Functional blockade of either degradative system leads to an enhanced aggresome formation. The tuberous sclerosis complex-Ras homologue enriched in brain-mammalian target of rapamycin (TSC-Rheb-mTOR) pathway is known to play a central role in modulating protein synthesis and autophagy. However, in spite of the constitutive activation of mTOR and the abrogated autophagy activity in TSC1- or TSC2-deficient cells, the TSC mutant cells are defective in aggresome formation and undergo apoptosis upon misfolded protein accumulation both in vitro and in vivo. High Rheb activity in TSC mutant cells inhibits aggresome formation and sensitizes cell death in response to misfolded proteins. Surprisingly, this previously unrecognized function of Rheb is independent of TOR complex 1. Active Rheb disrupts the interaction between dynein and misfolded protein cargos, and therefore blocks aggresome formation by inhibiting dynein-dependent transportation of misfolded proteins. This study reveals a function of Rheb in controlling misfolded protein metabolism by modulating aggresome formation.

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 inactivating 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 rapamycin (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.

Rapamycin-Insensitive Up-Regulation of MMP2 and Other Genes in Tuberous Sclerosis Complex 2–Deficient Lymphangioleiomyomatosis-Like Cells

Increased matrix metalloproteinase (MMP) activity has been implicated in the pathogenesis of lymphangioleiomyomatosis (LAM). The objective of this study was to investigate how tuberous sclerosis complex (TSC) 1 or TSC2 deficiency alters MMP expression and regulation. We studied immortalized cells that lack TSC2 derived from an angiomyolipoma of a patient with LAM, a TSC2 addback derivative, and murine embryonic fibroblast cells that lack Tsc1 or -2 and respective controls. Global gene expression analysis was performed in the angiomyolipoma and derivative cell lines. MMP levels in the conditioned media from these cells were analyzed by zymography and ELISA. We found increased MMP-2 expression in cells lacking TSC1/TSC2 compared with their respective controls by zymography. MMP-2 overproduction by these cells was not affected by rapamycin treatment. Gene expression analysis confirmed increased MMP-2 gene expression that was not affected by rapamycin. Furthermore, multiple other genes were found to be overexpressed in rapamycin-treated TSC2-deficient cells compared with TSC2(+) cells. We conclude that TSC1/TSC2 deficiency leads to MMP-2 overproduction that is rapamycin-insensitive, and that several genes exhibit similar patterns, suggesting that TSC1/TSC2-dependent, but mammalian target of rapamycin-independent, pathways may be involved in the pathogenesis of LAM.

AMPK Phosphorylation of Raptor Mediates a Metabolic Checkpoint

AMPK is a highly conserved sensor of cellular energy status that is activated under conditions of low intracellular ATP. AMPK responds to energy stress by suppressing cell growth and biosynthetic processes, in part through its inhibition of the rapamycin-sensitive mTOR (mTORC1) pathway. AMPK phosphorylation of the TSC2 tumor suppressor contributes to suppression of mTORC1; however, TSC2-deficient cells remain responsive to energy stress. Using a proteomic and bioinformatics approach, we sought to identify additional substrates of AMPK that mediate its effects on growth control. We report here that AMPK directly phosphorylates the mTOR binding partner raptor on two well-conserved serine residues, and this phosphorylation induces 14-3-3 binding to raptor. The phosphorylation of raptor by AMPK is required for the inhibition of mTORC1 and cell-cycle arrest induced by energy stress. These findings uncover a conserved effector of AMPK that mediates its role as a metabolic checkpoint coordinating cell growth with energy status.

Unfolding Possible Cancer Target

Work from Ozcan et al . weaves the unfolded protein response (UPR) [which is initiated in the endoplasmic reticulum (ER) when the capacity of the ER to produce properly folded proteins is exceeded] into a regulatory network regulating insulin signaling and apoptosis. The paper focuses on signaling through the mTORC1 complex, which regulates protein synthesis by controlling the activity of ribosomal protein S6 kinases and eukaryotic initiation factor 4E. Deletion or loss of function of the tuberous sclerosis complex genes (TSC1 and TSC2) leads to sustained activation of mTORC1. In mouse embryo fibroblasts lacking TSC1 and TSC2 or in tumor cells from Tsc2 +/– mice, the UPR response was activated. This UPR-dependent activation of the mitogen-activated protein kinase JNK and serine/threonine phosphorylation of insulin receptor substrate 1 provided a mechanism by which insulin receptor signaling was inhibited. Treatments that alleviated ER stress (such as the addition of a chemical chaperone that promoted proper protein folding) alleviated the defects in insulin signaling in Tsc1 –/– or Tsc2 –/– cells. When ER stress gets really out of hand, cells undergo apoptosis, and the authors showed that TSC-deficient cells had increased vulnerability to apoptosis when treated with chemical agents that enhance ER stress. The authors and Reiling and Sabatini in associated commentary discuss the appealing logic of having the UPR feed back onto the mTORC1 pathway, which responds to nutrients and regulates growth and proliferation, thus coordinating pathways regulating metabolism and survival. They further point out the possibility that the UPR may provide a target to promote elimination of tumor cells, specifically those cells lacking TSC proteins, or more generally in tumor cells whose rapid growth and high rates of protein synthesis might make them more vulnerable than normal cells. U. Ozcan, L. Ozcan, E. Yilmaz, K. Düvel, M. Sahin, B. D. Manning, G. S. Hotamisligil, Loss of the tuberous sclerosis complex tumor suppressors triggers the unfolded protein response to regulate insulin signaling and apoptosis. Mol. Cell 29 , 541-551 (2008). [PubMed] J. H. Reiling, D. M. Sabatini, Increased mTORC1 signaling UPRegulates stress. Mol. Cell 29 , 533-535 (2008). [PubMed]

Differential effects of energy stress on AMPK phosphorylation and apoptosis in experimental brain tumor and normal brain

BackgroundAMP-activated protein kinase (AMPK) is a known physiological cellular energy sensor and becomes phosphorylated at Thr-172 in response to changes in cellular ATP levels. Activated AMPK acts as either an inducer or suppressor of apoptosis depending on the severity of energy stress and the presence or absence of certain functional tumor suppressor genes.ResultsHere we show that energy stress differentially affects AMPK phosphorylation and cell-death in brain tumor tissue and in tissue from contra-lateral normal brain. We compared TSC2 deficient CT-2A mouse astrocytoma cells with syngeneic normal astrocytes that were grown under identical condition in vitro. Energy stress induced by glucose withdrawal or addition of 2-deoxyglucose caused more ATP depletion, AMPK phosphorylation and apoptosis in CT-2A cells than in the normal astrocytes. Under normal energy conditions pharmacological stimulation of AMPK caused apoptosis in CT-2A cells but not in astrocytes. TSC2 siRNA treated astrocytes are hypersensitive to apoptosis induced by energy stress compared to control cells. AMPK phosphorylation and apoptosis were also greater in the CT-2A tumor tissue than in the normal brain tissue following implementation of dietary energy restriction. Inefficient mTOR and TSC2 signaling, downstream of AMPK, is responsible for CT-2A cell-death, while functional LKB1 may protect normal brain cells under energy stress.ConclusionTogether these data demonstrates that AMPK phosphorylation induces apoptosis in mouse astrocytoma but may protect normal brain cells from apoptosis under similar energy stress condition. Therefore, using activator of AMPK along with glycolysis inhibitor could be a potential therapeutic approach for TSC2 deficient human malignant astrocytoma.

S6K1 Regulates GSK3 under Conditions of mTOR-Dependent Feedback Inhibition of Akt

Feedback inhibition of the PI3K-Akt pathway by the mammalian target of rapamycin complex 1 (mTORC1) has emerged as an important signaling event in tumor syndromes, cancer, and insulin resistance. Cells lacking the tuberous sclerosis complex (TSC) gene products are a model for this feedback regulation. We find that, despite Akt attenuation, the Akt substrate GSK3 is constitutively phosphorylated in cells and tumors lacking TSC1 or TSC2. In these settings, GSK3 phosphorylation is sensitive to mTORC1 inhibition by rapamycin or amino acid withdrawal, and GSK3 becomes a direct target of S6K1. This aberrant phosphorylation leads to decreased GSK3 activity and phosphorylation of downstream substrates and contributes to the growth-factor-independent proliferation of TSC-deficient cells. We find that GSK3 can also be regulated downstream of mTORC1 in a HepG2 model of cellular insulin resistance. Therefore, we define conditions in which S6K1, rather than Akt, is the predominant GSK3 regulatory kinase.

A Parallel Path to TOR

The mammalian target of rapamycin (mTOR) signaling pathway, which regulates protein synthesis and thereby cell growth and proliferation, is modulated both by amino acid availability and by insulin. Insulin regulates mTOR activity through a pathway that involves activation of class 1 phosphatidylinositol 3-kinase (PI3K) and the subsequent activation of PKB (protein kinase B), which phosphorylates and inactivates the tuberous sclerosis complex proteins 1 and 2 complex (TSC1/2), enabling the small GTPase (guanosine triphosphatase) Rheb to stimulate TOR signaling. It has not been clear, however, how information about nutrient supply regulates mTOR signaling. Nobukuni et al. found that, whereas amino acids, unlike insulin, had no effect on TSC2 phosphorylation on serine 939 (TSC2 S939), TSC2 abundance, or PKB activation, they stimulated phosphorylation of S6K1 (S6 kinase 1, a substrate downstream of mTOR) T389. Further, amino acid depletion inhibited S6K1 phosphorylation even in TSC2-deficient cells with enhanced Rheb activation. Although the PI3K inhibitor wortmannin inhibited amino acid-stimulated S6K1 phosphorylation, as did rapamycin, siRNA against class 1 PI3Kα did not. Amino acids stimulated the in vitro activity of immunoprecipitated class 3 PI3K hVps34 and the abundance in mouse embryo fibroblasts of its product, phosphatidylinositol 3-phosphate (PI3P). HEK293 cells transfected with GST-epitope-tagged hVps34 showed a dose-dependent increase in amino acid-mediated S6K1 phosphorylation, whereas hVps34 knockdown decreased this response, as did knockdown of hVps15 (which associates with and stimulates hVps34) or expression of a construct that competes for PI3P docking sites. Thus, the authors conclude that amino acids regulate TOR signaling through a pathway involving hVps34 that is parallel to that activated by insulin. T. Nobukuni, M. Joaquin, M. Roccio, S. G. Dann, S. Y. Kim, P. Gulati, M. P. Byfield, J. M. Backer, F. Natt, J. L. Bos, F. J. T. Zwartkruis, G. Thomas, Amino acids mediate mTOR/raptor signaling through activation of class 3 phosphatidylinositol 3OH-kinase. Proc. Natl. Acad. Sci. U.S.A. 102 , 14238-14243 (2005). [Abstract] [Full Text]

Farnesyltransferase inhibitors reverse altered growth and distribution of actin filaments in Tsc-deficient cells via inhibition of both rapamycin-sensitive and -insensitive pathways

Farnesyltransferase inhibitors (FTI) have been developed as anticancer drugs and are currently being evaluated in clinical trials. In this study, we have examined the effects of FTIs on Tsc-null cells to gain insight into their effects on farnesylated Rheb GTPase. This protein is involved in the activation of mTOR/S6K signaling and is down-regulated by the Tsc1/Tsc2 complex. Both Tsc1(-/-) and Tsc2(-/-) mouse embryonic fibroblasts exhibit constitutive activation of S6K and grow in the absence of serum. Two different FTI compounds, the clinical compound BMS-214662 and the newly described BMS-225975, inhibit the constitutive activation of mTOR/S6K signaling and block serum-free growth of the Tsc-null mouse embryonic fibroblasts. We have also found that Tsc-null mouse embryonic fibroblasts grow under anchorage-independent conditions and that both FTI compounds inhibit this soft agar growth. These FTI effects are similar to those observed with rapamycin. Another interesting phenotype of Tsc-null mouse embryonic fibroblasts is that they are round and contain actin filaments predominantly at the cell periphery. The addition of FTIs, but not rapamycin, led to the reappearance of intracellular actin filaments and reduction of peripheral actin filaments. The ability of FTI to rearrange actin filaments seems to be largely mediated by the inhibition of Rheb protein, as induction of intracellular actin filaments by FTI was much less efficient in Tsc2-null cells expressing Rheb (M184L), a geranylgeranylated mutant Rheb that can bypass farnesylation. These results reveal that FTIs inhibit Rheb, causing two different effects in Tsc-deficient cells, one on growth and the other on actin filament distribution.

Regulation of mTOR function in response to hypoxia by REDD1 and the TSC1/TSC2 tumor suppressor complex

Mammalian target of rapamycin (mTOR) is a central regulator of protein synthesis whose activity is modulated by a variety of signals. Energy depletion and hypoxia result in mTOR inhibition. While energy depletion inhibits mTOR through a process involving the activation of AMP-activated protein kinase (AMPK) by LKB1 and subsequent phosphorylation of TSC2, the mechanism of mTOR inhibition by hypoxia is not known. Here we show that mTOR inhibition by hypoxia requires the TSC1/TSC2 tumor suppressor complex and the hypoxia-inducible gene REDD1/RTP801. Disruption of the TSC1/TSC2 complex through loss of TSC1 or TSC2 blocks the effects of hypoxia on mTOR, as measured by changes in the mTOR targets S6K and 4E-BP1, and results in abnormal accumulation of Hypoxia-inducible factor (HIF). In contrast to energy depletion, mTOR inhibition by hypoxia does not require AMPK or LKB1. Down-regulation of mTOR activity by hypoxia requires de novo mRNA synthesis and correlates with increased expression of the hypoxia-inducible REDD1 gene. Disruption of REDD1 abrogates the hypoxia-induced inhibition of mTOR, and REDD1 overexpression is sufficient to down-regulate S6K phosphorylation in a TSC1/TSC2-dependent manner. Inhibition of mTOR function by hypoxia is likely to be important for tumor suppression as TSC2-deficient cells maintain abnormally high levels of cell proliferation under hypoxia.

Tuberous sclerosis complex tumor suppressor-mediated S6 kinase inhibition by phosphatidylinositide-3-OH kinase is mTOR independent.

The evolution of mitogenic pathways has led to the parallel requirement for negative control mechanisms, which prevent aberrant growth and the development of cancer. Principally, such negative control mechanisms are represented by tumor suppressor genes, which normally act to constrain cell proliferation (Macleod, K. 2000. Curr. Opin. Genet. Dev. 10:81–93). Tuberous sclerosis complex (TSC) is an autosomal-dominant genetic disorder, characterized by mutations in either TSC1 or TSC2, whose gene products hamartin (TSC1) and tuberin (TSC2) constitute a putative tumor suppressor complex (TSC1-2; van Slegtenhorst, M., M. Nellist, B. Nagelkerken, J. Cheadle, R. Snell, A. van den Ouweland, A. Reuser, J. Sampson, D. Halley, and P. van der Sluijs. 1998. Hum. Mol. Genet. 7:1053–1057). Little is known with regard to the oncogenic target of TSC1-2, however recent genetic studies in Drosophila have shown that S6 kinase (S6K) is epistatically dominant to TSC1-2 (Tapon, N., N. Ito, B.J. Dickson, J.E. Treisman, and I.K. Hariharan. 2001. Cell. 105:345–355; Potter, C.J., H. Huang, and T. Xu. 2001. Cell. 105:357–368). Here we show that loss of TSC2 function in mammalian cells leads to constitutive S6K1 activation, whereas ectopic expression of TSC1-2 blocks this response. Although activation of wild-type S6K1 and cell proliferation in TSC2-deficient cells is dependent on the mammalian target of rapamycin (mTOR), by using an S6K1 variant (GST-ΔC-S6K1), which is uncoupled from mTOR signaling, we demonstrate that TSC1-2 does not inhibit S6K1 via mTOR. Instead, we show by using wortmannin and dominant interfering alleles of phosphatidylinositide-3-OH kinase (PI3K) that increased S6K1 activation is contingent upon the suppression of TSC2 function by PI3K in normal cells and is PI3K independent in TSC2-deficient cells.

Enhanced cardiomyocyte DNA synthesis during myocardial hypertrophy in mice expressing a modified TSC2 transgene.

Tuberous sclerosis complex (TSC) is a rare genetic disorder characterized by the appearance of benign tumors in multiple organs, including the heart. Disease progression is accompanied by homozygous mutation at 1 of 2 loci (designated TSC1 or TSC2), leading to the suggestion that these genes function as tumor suppressors. In this study, we generated a series of TSC2 cDNAs in which one or more structural motifs were deleted, with the hope that expression of the modified gene product would override the growth-inhibitory activity of the endogenous TSC2 gene product. Several of the modified cDNAs enhanced growth rate, increased endocytosis, and promoted aberrant protein trafficking when expressed in NIH-3T3 cells, thereby mimicking phenotypes typical of TSC2-deficient cells. Surprisingly, targeted expression of the most potent TSC2 cDNA to the heart did not perturb cardiac development. However, the level of cardiomyocyte DNA synthesis in adult transgenic mice was elevated >35-fold during isoproterenol-induced hypertrophy compared with their nontransgenic siblings. These results suggest that alteration of TSC2 gene activity in combination with beta-adrenergic stimulation can reactivate the cell cycle in a limited number of terminally differentiated adult cardiomyocytes.