La croissance des cellules est au cœur de nombreuses pathologies humaines. C’est l’un des aspects les plus fondamentaux du devenir des cellules, avec la prolifération et la mort cellulaire.
Participants
Joseph Avruch, John Blenis, Lewis Cantley, Karen Cichowski, Peter Devreotes, Eyal Gottlieb, Kun-Liang Guan, Michael N. Hall (organisateur), Peter J. Houghton, Brian K. Kennedy, Pierre Leopold, Alison Lloyd, Brendan Manning, Christian Meyer, Sergio Moreno, Thomas P. Neufeld, Linda Partridge, Mario Pende, David Sabatini (organisateur), Barbara Marte (éditrice)
Résumé en français
La croissance des cellules est au cœur de nombreuses pathologies humaines. C’est, avec la prolifération et la mort cellulaire, un des aspects les plus fondamentaux du devenir des cellules. La croissance cellulaire fixe non seulement la taille et la forme des cellules individuelles, mais également la taille des tissus et des organismes.
Des dysfonctionnements dans la voie de la kinase TOR qui contrôle la croissance cellulaire produisent des défauts de développement ainsi qu’une série de pathologies majeures telles que cancers, diabète et obésité. Si l’on en croit la progression exponentielle du nombre de publications scientifiques, le domaine d’étude de la voie TOR est en rapide expansion et englobe un nombre croissant de domaines associés comme, récemment, le vieillissement. La conférence « Cell growth and the TOR pathway » organisée par la fondation Les Treilles en juin 2008 a rassemblé 20 scientifiques de six pays différents ainsi qu’un éditeur de la revue Nature pour discuter les avancées les plus récentes dans le domaine de la croissance et de la biologie de la kinase TOR. Plusieurs aspects ont été abordés au cours des différentes présentations, tels que la fonction de la signalisation TOR dans les différents tissus des organismes complexes, le lien entre la signalisation TOR et les acides aminés, l’interface entre TOR et le métabolisme cellulaire, ainsi que le rôle de la signalisation TOR dans le vieillissement et les maladies liées à l’âge. Reflet du haut niveau de conservation moléculaire et de l’importance des mécanismes de croissance, les recherches présentées ont porté sur des modèles biologiques très variés tels que levures, plantes, vers, insectes, souris et homme. La conférence s’est conclue sur une discussion abordant les voies à suivre pour le futur, ainsi que les questions qui restent pour le moment sans réponse.
Compte rendu
Mike Hall started the meeting and his talk with a global overview of TOR signaling. TOR (target of rapamycin) is a highly conserved serine/threonine kinase that controls cell growth and metabolism in response to nutrients, growth factors, cellular energy, and stress. TOR was originally discovered in yeast but is conserved in all eukaryotes including plants, worms, flies, and mammals. The discovery of TOR led to a fundamental change in how one thinks of cell growth. It is not a spontaneous process that just happens when building blocks (nutrients) are available, but rather a highly regulated, plastic process controlled by TOR-dependent signalling pathways. TOR is found in two structurally and functionally distinct multiprotein complexes, TORC1 and TORC2. The two TOR complexes, like TOR itself, are highly conserved. Thus, the two TOR complexes constitute an ancestral signalling network conserved throughout eukaryotic evolution to control the fundamental process of cell growth. As a central controller of cell growth, TOR plays a key role in development and aging, and is implicated in disorders such as cancer, cardiovascular disease, obesity, and diabetes.
Next, Hall discussed the effects of knocking out TORC1 and TORC2 signaling in specific tissues. While the role of TOR in controlling growth of single cells is relatively well understood, the challenge now is to understand the role of TOR signaling in coordinating and integrating overall body growth in multicellular organisms. This will require elucidating the role of TOR signaling in individual tissues. He presented data on the roles of mTORC1 and mTORC2 in adipose tissue and in muscle. In particular, he presented evidence that adipose mTORC1 is a regulator of adipose metabolism and thereby controls whole body energy homeostasis. Adipose mTORC2 plays an unexpectedly central role in controlling whole body growth. Studies performed in collaboration with Markus Rüegg demonstrated that muscle-specific ablation of mTORC1causes muscle dystrophy, whereas loss of mTORC2 in the muscle has little-to-no effect.
Finally, Hall addressed the issue of TOR localization in cells. There are several studies published describing TOR localization, but with seemingly contradictory results. The inconsistent results might be due to the fact that all published studies relied on fixed or broken cells. Hall described experiments on localization of functional, GFP-tagged TOR in live yeast cells, concluding that TOR has multiple, distinct locations.
David Sabatini (Whitehead Institute/MIT, Cambridge, MA, USA) continued on the theme of TOR signaling with a presentation on the regulation of the mTORC1 pathway by amino acids. He began with a very brief review of the mammalian TOR pathway and focused on the identification of Rag GTPases as important mediators of the amino acid input to mTORC1. He showed data indicating the a heterodimer of the Rags interacts with mTORC1 via its raptor component in a GTP dependent fashion. Moreover, both the GTP loaded state of the Rags as well their interaction with mTORC1 are regulated by amino acids. Expression in cells of a Rag mutant that is constitutively bound to GTP leads to the activation of the mTORC1 pathway as well as makes it insensitive to amino acid deprivation. Conversely, expression of Rag mutant that cannot bind GTP inhibits the mTORC1 pathway so that it cannot be activated by any upstream signal.
Dr. Sabatini then discussed potential mechanisms through which the Rag proteins my act and said that the simple model where Rag binding to mTORC1 causes activation of the mTORC1 kinase activity does not appear to be true. Although the addition of GTP-loaded Rheb to mTORC1 causes a great increase in mTORC1 kinase activity the addition of analogous preparations of the Rags has no affect on kinase activity. Instead, Dr. Sabatini presented evidence suggesting the Rag GTPases control the intracellular localization of mTORC1. Stimulation of mammalian cells with amino acids leads to the translocation of mTORC1 from a punctate cytoplasmic location to cellular membranes that are positive for the late endosomal/lysosomal marker rab7. Interestingly, expression in cells of a the Rag mutant that is constitutively bound to GTP mimics the effects of amino acids on mTORC1 location while equivalent experiments with Rheb does not have this effect. While the intracellular location of Rheb does not change with amino acids, Dr. Sabatini proposed that the Rag-mediated of mTORC1 to the Rab7 positive comparment leads to its co-localization with Rheb, which previous workers have shown co-localizes with Rab7. Thus, amino acids, in a Rag-dependent mechanism, cause the movement of mTORC1 to the same compartment that contains its activator, Rheb.
The second half of his presentation was dedicated to describing results showing that mTORC2—the PDK2 kinase for Akt/PKB—is necessary for the formation of prostate tumors caused by the loss of the tumor suppressor PTEN. Interestingly, mTORC2 does not appear to be necessary for the normal function of the prostate, suggesting mTORC2 may be an interesting drug target for cancer therapy.
John Blenis (Harvard Medical School, Boston, MA, USA) focused his lecture on the regulation of protein synthesis and cell growth by mTORC1 and its effectors, the S6 kinases and 4EBP1/eIF4E. His results begin to provide a molecular understanding of how these signaling proteins interface with the translational machinery in a complex but ordered fashion to coordinate a protein synthetic growth response to nutrient and energy sufficiency and mitogens. In particular, his data provide new insights into how mTORC1 increases steady state translational efficiency of mRNAs possessing highly structured 5’UTRs encoding critical regulators of cell cycle progression, bioenergetics and angiogenesis. He also presented new data describing how mTORC1 and S6K1 increase the overall efficiency of translation on newly spliced mRNA through the recruitment of activated S6K1 and SKAR to exon junction complexes formed on newly synthesized and spliced mRNA. Since the first round of translation, referred to as the Pioneer round, is uniquely positioned to act as a decision point for the cell to decide whether to invest in the energy consuming process of steady state translation, John proposed that this process is an important energy and nutrient sensing checkpoint for cell growth control. Finally, John presented work-in-progress investigating why the mTORC1 inhibitor rapamycin, exhibits differential effects on suppression of steady state translation is various cell types. In cells that are less sensitive to rapamycin-mediated inhibition of steady state translation, it appears that chronic treatment of rapamycin maintains S6 kinases in the inactive state while 4EBP1 phosphorylation becomes rapamycin resistant. Although the molecular mechanism is still unclear, these results have important implications with regard to the use of rapamycin in the clinic.
Brendan Manning (Harvard School of Public Health, Boston, MA, USA) presented data on three new stories related to regulation and function of the two mTOR complexes. A novel approach to define mTORC1-regulated transcripts was described. This approach combines loss of the TSC1-TSC2 complex to activate mTORC1 with a time course of rapamycin treatment to inhibit mTORC1. Using stringent statistical criteria, 239 genes whose expression levels are most strongly affected by mTORC1 activation were defined. Of these, 152 were induced by mTORC1 activity, while 87 were repressed. Gene-set enrichment analysis of the 152 mTORC1-induced transcripts demonstrated an over-representation of genes involved metabolic processes (glycolysis, pentose phosphate pathway, lipid and sterol biosynthesis) and the function of specific cellular organelles (endoplasmic reticulum (ER), lysosome, mitochondria). Twenty-five previously characterized targets of the transcription factor hypoxia-inducible factor (HIF) were found as being mTORC1 induced, and HIF1alpha levels were increased in a number of in vitro and in vivo settings with elevated mTORC1 signaling. In a second story, Brendan showed data demonstrating that aberrantly high mTORC1 signaling causes ER stress and activates the unfolded protein response in cells and tumors lacking the TSC tumor suppressors. The activation of this stress response pathway offers a potential therapeutic opportunity to selectively kill tumor cells that have elevated mTORC1 activity, which is common in human tumor syndromes and cancer. Finally, an update on a very recently published story from the Manning lab regarding regulation of mTORC2 by the TSC1-TSC2 complex was presented. These findings demonstrate that mTORC2 activity is impaired upon disruption of the TSC1-TSC2 complex and that this effect can be separated from well-characterized effects on mTORC1. Collectively, the data presented suggest that the TSC1-TSC2 complex inhibits mTORC1, while activating mTORC2. The molecular mechanism of this activation is being further explored. However, it appears to be related to a physical, albeit transient, interaction between the TSC1-TSC2 complex and mTORC2, rather than the previously known function of the TSC1-TSC2 complex serving as a GAP for Rheb.
Joseph Avruch (Massachusetts General Hospital, Boston MA, USA) continued the session by focusing on the role of PRAS40 in regulation of mTORC1. He showed that PRAS40 is a substrate for the mTORC1 kinase activity and that this phosphorylation is necessary for de-repression of mTORC1 by upstream signals. He stressed that PRAS40 is a substrate for mTORC1, just like S6K1 and 4E-BP1 and that perhaps its inhibitory properties are a result of competition with other subtrates. He showed that Serine183 of PRAS40 is the predominant mTORC1 phosphorylation site and that it is regulated like standard mTORC1 sites. He also discussed the identification of several phosphorylation sites on raptor whose mutation does not affect raptor binding to 4E-BP1. Dr. Avruch also briefly discussed the identification of myo1c as a rictor binding protein and cautioned that mTOR complex components, like rictor, may have mTOR independent roles.
In the last talk of the opening day Kun-Liang Guan (University of California San Diego, CA, USA) presented two new studies on the target of rapamycin (TOR) complexes, TORC1 and TORC2. TORC1 plays a critical role in the regulation of cell growth and cell size. A wide range of signals, including amino acids, is known to activate TORC1. Research from Dr. Guan and colleagues have identified the Rag GTPases, which are members of the Ras superfamily, as novel activators of TORC1 in response to amino acid signals. Knockdown of Rag gene expression suppressed the stimulatory effect of amino acids on TORC1 in Drosophila S2 cells. Expression of constitutively active (GTP-bound) Rag in mammalian cells enhances TORC1 in the absence of amino acids while expression of dominant negative Rag blocks the stimulatory effects of amino acids on TORC1. In collaboration with Dr. Tom Neufeld, Drosophilagenetic studies also show that the Rag GTPases regulate cell growth, autophagy, and animal viability under starvation. Together, these studies establish a novel function of Rag GTPases in TORC1 activation in response to amino acid signals. Dr. Guan also discussed the function of TORC2 in phosphorylation and stability regulation of protein kinase C, PKC, which is involved in a wide array of cellular processes such as cell proliferation, differentiation, and apoptosis. Phosphorylation of both turn motif (TM) and hydrophobic motif (HM) are important for PKC function. Their studies show thatTORC2 plays a pivotal role in phosphorylation of both TM and HM in all conventional PKCs, novel PKCε, as well as Akt. Ablation of mTORC2 components (Rictor, Sin1, or mTOR) abolished TM phosphorylation and decreased HM phosphorylation of PKCα. Interestingly, the mTORC2-dependent TM phosphorylation is essential for PKCα maturation, stability, and signaling. These observations demonstrate that TORC2 is involved in posttranslational processing of PKC by facilitating TM and HM phosphorylation, and reveals a novel function of TORC2 in cellular regulation
On the morning of June 4th Sergio Moreno (CSIC/Universidad de Salamanca, Salamanca, Spain) described that in fission yeast the TOR pathway controls cell growth and cell differentiation, such that they become mutually exclusive. Fission yeast Tor2 regulates cell growth, controls ribosome biogenesis, and associates with the Raptor homologue Mip1, forming the growth controlling TORC1 complex. Moreover, Tor2 has an additional and unexpected function in repressing sexual differentiation. Accordingly, Tor2 overexpression strongly reduces meiosis and sporulation efficiency while Tor2 inactivation has the opposite effect, leading to G1 arrest and to cell differentiation, mating and meiosis, regardless of the nutritional conditions. This new function of Tor2 seems to operate by interfering with the functions of the transcription factor Ste11 and the meiosis-promoting RNA binding protein Mei2. Therefore, in fission yeast TOR is a key regulator of the switch between cell growth and cell differentiation in response to nutrient availability.
Tony Hunter (UCSD, San Diego, CA, USA) continued the focus on TOR signaling by describing that pathways are commonly drawn as linear unidirectional pathways. However, genetic knockout analysis and the use of selective protein kinase inhibitors is increasingly revealing feedback loops within phosphorylation signaling pathways and also unexpected crosstalk between pathways. Hunter reported that the mTOR pathway is exquisitely regulated through such mechanisms in mammalian cells. The activity of the mTORC1 complex, a key regulator of both growth factor and nutrient signaling via S6 kinase (S6K), is modulated by a negative feedback loop in which S6K, activated downstream of mTORC1, phosphorylates the IRS1 and IRS2 adaptor proteins, uncoupling them from activated insulin/IGF-1 receptor tyrosine kinases, and also inducing their downregulation at the protein level, with the net result that there is reduced PI-3K signaling to Akt and decreased mTORC1 activity. Activated mTOR also negatively regulates NF-B induction by TNF and DNA damage, through crosstalk to the ERK and PI-3K pathways. Another potential negative feedback loop involves activated Akt, whose activity somehow reduces activating phosphorylation of Akt at S473 by mTORC2 a complex that regulates the actin cytoskeleton and several AGC family kinases through hydrophobic motif phosphorylation. In studies of how mTORC1 and mTORC2 might be regulated by differential phosphorylation, Hunter reported that mTOR is phosphorylated differentially when associated with mTORC1 and mTORC2, when isolated from 293 and U2OS cells stimulated with insulin or IGF-1; mTORC1 contained mTOR phosphorylated predominantly on Ser2448, whereas mTORC2 contained mTOR phosphorylated predominantly on Ser2481. Phosphorylation of both sites required intact mTOR complexes. S2481 could be a mTORC2-dependent autophosphorylation site, perhaps dependent on mTORC2 dimerization, whereas S2448 is a known S6K site, and could be phosphorylated as another feedback response. Prolonged rapamycin treatment, which induces mTORC2 disassembly, reduced mTOR pS2481 levels. Using S2481 phosphorylation as a marker for mTORC2 sensitivity to rapamycin, mTORC2 was found to be rapamycin sensitive in several cancer cell lines in which it had been previously reported that mTORC2 activity was rapamycin insensitive, based on the level of Akt S473 phosphorylation (e.g. MDA-MB-468 cells). Based on Rictor or mSin1 depletion, it appears that residual mTORC2 activity in these cells is primarily responsible for rapamycin-resistant S473 phosphorylation, although there may also be a contribution from DNA-PK, which, like mTOR, is a member of the PIKK family of atypical protein kinases.
Peter Devreotes (The Johns Hopkins Medical School, Baltimore, MD, USA) discussed the role of Tor complex 2 (TorC2) in chemotaxis. He explained that high levels of phosphotidylinositol 3, 4, 5 tris phosphate (PIP3) as occur in cells lacking PTEN, cause cytoskeletal rearrangements and alter cell motility and chemotaxis. However, chemotaxis can still occur in the absence of PIP3 , indicating there are PIP3-independent pathways. He outlined a PIP3-independent pathway linking temporal and spatial activation of PKBs by TorC2 to the chemotactic response. Within seconds of stimulating Dictyostelium cells with chemoattractant, two PKB homologs, PKBA and PKBR1, mediate transient phosphorylation of at least eight proteins, including Talin, PI4P 5-kinase, two RasGefs, and a RhoGap. Surprisingly, all of these substrates are phosphorylated with normal kinetics in cells lacking PI 3-kinase activity. However, cells deficient in TorC2 or PKBR1 activity show reduced phosphorylation of the substrates and are impaired in chemotaxis. The PKBs are activated through phosphorylation of their hydrophobic motifs via TorC2 and subsequent phosphorylation of their activation loops. For PKBR1, these chemoattractant-inducible events are restricted to the cell’s leading edge even in the absence of PIP3. Activation of TorC2 depends on heterotrimeric G-protein function and intermediate G-proteins, including Ras GTPases. Devreotes presented a model where cytosolic TorC2, encountering locally activated small G-protein(s) at the leading of the cell, becomes activated and phosphorylates PKBs. These in turn phosphorylate a series of signaling and cytoskeletal proteins, thereby regulating directed migration.
Lewis Cantley (Beth Israel Hospital, Boston, MA, USA) focused on the role of PI3K in cancer as well as on cancer cell metabolism. He discussed new results showing that expression of a mutant allele of PI3K found in human tumors leads to the formation of lung tumors in mice. Interestingly, treatment of the mice with an experimental drug that inhibits PI3K led to the complete regression of the tumors. On the other hand, established lung tumors caused by the expression of a constitutively active allele of ras are not affected by the same PI3K inhibitor. However, if the inhibitor is given before the onset of tumor formation it does block tumorigenesis. These results suggest that PI3K activity is needed to maintain tumors caused by hyperactive PI3K but not ras signaling but that ras-driven tumors do require PI3K for the initial stages of tumor formation. The PI3K inhibitor also dramatically inhibits glucose uptake into tumors in vivo.
In the second half of his lecture, Dr. Cantley focused on the essential role of pyruvate kinase M2 (PKM2) in cancer cell proliferation. PKM2 is a splice form of pyruvate kinase that is expressed during embryogenesis and apparently in all dividing cells as well as adipocytes. Using loss of function experiments, Dr. Cantley showed that human cancer cells cannot form tumors in vivo if they lack PKM2. Moreover, PKM2 but not PKM1, is regulated by tyrosine kinase signaling.
After the coffee break Eyal Gottlieb (Beatson Institute for Cancer Research, Glasgow, Scotland) discussed the role of prolyl hydroxylase enzymes in metabolic sensing and cancer. The survival of a cell under low oxygen (hypoxia) is dependent on its ability to adapt its metabolism. Hypoxic stress is particularly important in cancer development, where cells are frequently located too far from functional blood vessels to allow for appropriate oxygenation. Hypoxia is a major selective pressure in forming aggressive tumors, therefore providing a strong rationale for development of drugs that target hypoxia regulatory pathways. HIF transcription factors direct the majority of gene expression under hypoxia, and are themselves upregulated in low oxygen via functional inactivation of HIF prolyl hydroxylase enzymes (PHDs). Eyal showed that by using esterified forms of a -ketoglutarate, a PHD substrate, reactivation of these enzymes under hypoxia destabilized HIF a, and reduced expression of its transcriptional targets. Eyal also showed that in doing so, hypoxia-induced glycolysis and ATP levels were decreased, and cell death was promoted. Finally, Eyal demonstrated that treatment of mice with a cell-permeable a -ketoglutarate derivative reduced xenograft tumor growth and decreased HIF activation. Eyal suggested that PHDs can be viewed as valid, druggable targets applicable to most solid tumors, and therefore their activation represents a novel approach to anticancer therapy.
Furthermore, Dr. Gottlieb has demonstrated that amino acids can transmit a signal to PHDs by regulating a-ketoglutarate levels in cells. Following amino acid deprivation, a -ketoglutarate levels are dramatically decreased leading to functional inactivation of PHDs. Eyal has provided evidence that this a-ketoglutarate-dependent regulation of PHDs is potentially an important step in the amino acid sensing machinery which leads to Tor activation. Eyal suggested that PHDs can serve not only as potent oxygen sensors but also as amino acid sensors in cells.
Alison Lloyd (University College London, London, UK) concluded the afternoon session by discussing how normal mammalian cells will only grow when instructed to do so by extracellular growth factors. Dr. Lloyd discussed results obtained using a cultured, primary cell system relating to (i) how extracellular growth factors and mitogens act to regulate cell size (ii) how oncogenes and tumour suppressors drive cell growth independently of normal growth controls (iii) the regulation of mitochondrial biogenesis in normal and cancer cells. The regulation of cell size in mammalian cells is a controversial topic. Using classical “switch” experiments between culture conditions that produce small or large cells, Lloyd presented evidence that extracellular factors set independent growth and proliferation rates that together determine cell size. The conclusions of the work argued against the existence of a “size checkpoint” and demonstrated that proliferation rates are unaffected by the size of the cell. Tumours result from loss of normal growth and proliferative controls. Lloyd showed a role for Rb in controlling cell growth and that, similarly to effects on the cell-cycle, oncogenes and tumour suppressors cooperate to drive strong, sustained growth in the absence of extracellular factors. Cells generate new organelles when stimulated to grow and divide; however, little is known about how growth and mitogenic signalling pathways regulate organelle biogenesis. Using mitochondria as a model organelle, Dr. Lloyd reported that ERK and PI3-kinase signalling pathways act synergistically to increase mitochondrial biogenesis and mitochondrial DNA replication, resulting in increased mitochondrial density in proliferating cells. Interestingly, this transcriptionally-driven process is independent of Akt/TOR signalling, a major regulator of cell growth in these cells. This separation of the pathways that drive mitochondrial biogenesis and cell growth provides a mechanism to modulate mitochondrial density according to the metabolic requirements of the cell.
As the first speaker on June 5, Mario Pende (Paris Descartes University Medical School, Paris, France) presented data on mouse models carrying mutations in the mTOR, Akt and S6 kinase genes to study the interaction between these signalling elements and their involvement in mammalian pathophysiology. To study mTOR function in differentiated tissues, the mTOR gene was conditionally deleted using a human skeletal actin promoter driven Cre (in collaboration with the Gangloff group, ENS, Lyon). Young mice are viable, though they develop kyphosis and abnormal posture. Mutant mice die at 6-7 months of age, possibly due to respiratory failure. As expected, mTOR mutant muscles are atrophic. However slow twitch muscles such as soleus and diaphragm also show signs of dystrophy and display pale colour, indicating that defects in the nutrient/mTOR pathway may contribute to muscle dystrophy and muitochondrial myopathy. Most of mTOR targets are down-regulated in the mutant muscle with the important exception of Akt that is compensatorily up-regulated. Defects in dystrophin expression and mitochondrial biogenesis may contribute to the phenotype of the mTOR mutant muscles. Mario Pende went on and discussed the genetic interaction between Akt and S6K mutants in mice. S6K1 deletion causes small cell size, insulin hypersensitivity and reduced insulin levels. S6K1 deficient muscle cells present with energy stress and AMP-activated kinase up-regulation. These changes are accompanied by metabolic adaptations leading to fatty acid b-oxidation and a sharp depletion of lipid content, while glycogen stores are spared. Constitutively active Akt1 (MyrAkt1) triggers mTOR signalling and S6K1 activity in a variety of tissues. MyrAkt1 expression in pancreatic b-cells up-regulates cell size and insulin production, though causes insulinoma formation in later life. S6K1 deletion is sufficient to completely block the hyperplasia stage of tumorigenesis in this mouse model of cancer. These results identify S6K1 as a critical element in mTOR signalling that may represent a useful target against metabolic diseases and cancer.
Peter Houghton (St. Jude Children’s Research Hospital, Memphis, TN, USA ) continued the focus on TOR and disease in his lecture. He discussed that mTORC1 signaling is negatively regulated by cellular stress such as DNA damage, nutritional deficiency and hypoxia. However, in many solid tumors mTORC1 signaling is maintained despite both suboptimal nutrition and hypoxia. Regulation of mTORC1 by hypoxia is mediated by hypoxia inducible factor 1a (HIF1a) induction of REDD1 and activation of the TSC complex. However, the signaling pathway upstream of HIF1a is poorly understood. Using murine embryo fibroblasts and human fibroblasts it was shown that inhibition of mTORC1 signaling under hypoxia required ATM. Cells deficient in ATM maintained mTORC1 signaling under conditions of DNA damage and hypoxia. Although under hypoxia HIF1a was stabilized it failed to induce REDD1 in the absence of ATM. ATM was found to phosphorylate HIF1a in vitro at S696. Expression of an ATM mutant (S696R) failed to support REDD1 induction under hypoxic conditions, suggesting that in vivo ATM phosphorylation of HIF1a is critical in control of mTORC1 signaling under hypoxic conditions of cell growth.
Previously it was shown that rapamycin induced apoptosis in p53-mutant sarcoma cells under growth factor deficient conditions. Only insulin-like growth factors (IGF-1, IGF-2) could rescue from rapamycin-induced cell death. In vitro the combination of rapamycin with an antibody that blocks ligand binding to the IGF-1 receptor (CP751871) significantly potentiated rapamycin-induced death. To investigate the therapeutic potential, mice bearing advanced subcutaneous implants of childhood Ewing sarcoma (n=6), osteosarcoma (n=4) or rhabdomyosarcoma (n=2) were treated with CP751871 twice weekly for 4 weeks, rapamycin daily for 5 days for up to 12 consecutive weeks, or the combination. The combination was synergistic or supra-additive in 6 of 12 models, inducing complete tumor regression in 1 Ewing, 1 rhabdomyosarcoma and 3 osteosarcoma models.
Karen Cichowski (Brigham & Women’s Hospital, Boston, MA, USA) next discussed the role of mTOR signaling and the therapeutic effects of mTOR inhibitors in Ras-driven tumors. For the majority of her talk she focused on tumors associated with the familial cancer syndrome neurofibromatosis type 1 (NF1). NF1 is a fairly common disease caused by loss of function mutations in the NF1tumor suppressor, a gene that encodes a RasGAP protein. Accordingly, aberrant Ras signaling underlies disease pathogenesis. However, until recently the specific effector pathways contributing to tumor development were unknown. Dr. Cichowski discussed her work showing that the NF1 tumor suppressor critically regulates the mTOR pathway via its effects on Ras, PI3K and TSC2. Importantly, she demonstrated that the mTOR pathway is hyper-activated aggressive nervous system sarcomas from NF1 patients and that tumor cell lines are exquisitely sensitive to mTOR inhibitors. Moreover, the mTOR inhibitor rapamycin potently suppressed the development of these sarcomas in a genetically engineered mouse tumor model. These data suggest that mTOR inhibitors may represent a potential therapy for these aggressive malignancies that are often refractory to standard chemotherapy.
Dr. Cichowski then discussed how she has been extending these studies to develop novel therapies for these NF1-asscoiated tumors and for other tumor types, including gliomas and lung cancer. First, she discussed the development of several new therapies, combining rapamycin with other agents, which are showing promise in pre-clinical studies in her mouse tumor model. In addition she has begun to explore the effects of these inhibitors in lung cancers that harbor K-Ras mutations. Interestingly, while K-Ras mutations alone do not confer sensitivity to rapamycin, tumors that harbor mutations in multiple genes that affect the mTOR pathway are sensitive to these agents in vitro and in vivo. She has begun to dissect the genetic determinants of this sensitivity and is currently investigating mechanisms of resistance.
The afternoon of June 5 was spent on an excursion to a nearby Abbey.
On the morning of June 6th, Thomas Neufeld (University of Minnesota, MI, USA) discussed the role of the Rag proteins in Drosophila as well as the identification of a fly version of ATG13. Loss of the Rag gene in cell clones in vivo leads to a reduction in cell size and an increase in autophagy, phenotypes consistent with dTORC1 inhibition. Moreover, these effects cannot be rescued by a concomitant deletion of TSC1/TSC2, suggesting that the Rag proteins act downstream of Rheb.
An important question in the TOR field is how TORC1 regulates autophagy in flies and mammalian cells. In yeast it is clear that TOR regulates the ATG1-ATG13 protein complex but no orthologue of ATG13 had been identified in flies or mammals. Dr. Neufeld presented evidence that his lab has identified a fly orthologue and that this protein is necessary for autophagy in vivo. Like ATG1, loss of ATG13 completely blocks induction of autophagy. Moreover, ATG13 phosphorylation appears to be regulated by TOR and ATG1 activity but not apparently the ATG1-ATG13 interaction. It remains to be seen if TOR directly mediates these phosphorylation events. Unexpectedly, S6K has a positive role in the control of autophagy.
In the second half of his talk, Dr. Neufeld discussed how vps34, a potential regulator of mTORC1 in mammals, does not regulate dTORC1 in flies in vivo. Loss of vps34 does not affect cell size nor does it block Rheb-induced cell growth. Cells lacking vps34 have a partial defect in endocytosis.
Pierre Leopold (Université de Nice, Nice, France) gave a presentation on the humoral control of growth in Drosophila. The control of growth at the level of a whole organism involves a series of intricate humoral regulations allowing the coordination of growth programs in all tissues. One key feature of this global control is to integrate extrinsic influences, like nutrition, as well as intrinsic mechanisms, which are linked to the program of development. Pierre Léopold and co-workers presented a series of experiments addressing these issues in the genetic model of the fruit fly Drosophila. These experiments suggest that nutrition is coupled with growth control through the function of a general sensor operating in the fat body of the animal. This specific organ combines the functions of the liver and the fat tissue of vertebrates. Léopold presented evidence that upon specific amino acid starvation, the sensor mechanism in this tissue operates through a downregulation of the TOR branch of the Insulin/IGF signalling pathway (IIS), and induces a remote inhibition of organismal growth via local repression of PI3-kinase signaling in peripheral tissues. The nature of this remote inhibition of IIS is not well understood, but several mechanisms could be involved, like a control of insulin/IGF secretion in the brain cells that produce it or a modification of its biological function through an association with binding proteins similar to the IGF-BPs and ALS. Whereas TOR in the fat body controls the growth rate, final organism size is also determined by the duration of the growth period. In a second part of his talk, Léopold presented evidence suggesting that the length of the growth period is controlled by TOR-mediated regulation of ecdysone production in the ring gland. These findings overall suggest that TOR could be a central controller of organismal growth through its participation to a general nutrition sensor, and that insulin/IGF signaling would respond to the function of this general sensor in peripheral tissues.
Linda Partridge (University College London, London, UK) discussed the role of nutrients and nutrient-sensing in ageing, particularly in the fruit fly Drosophila. Recent work has demonstrated that mutations in single genes can slow down the ageing process in laboratory model organisms, and both the insulin/Igf and TOR pathways have an evolutionarily conserved effect on ageing. Our knowledge of these signalling pathways is still incomplete, and recent unpublished work with Drosophila has shown that mutations in lnk, which encodes the single fly orthologue of mammalian Lnk, APS and SH2B, can extend the lifespan of the fly, reduce female fecundity, increase stress resistance and storage of carbohydrates and lipids. Mutants in the pathway that increase lifespan in the nematode worm Caenorhabditis elegans, Drosophila and the mouse elevate RNA transcripts of genes that function in phase 1 and 2 cellular detoxification, and recent experimental work has demonstrated that direct elevation of cellular detoxification extends lifespan in C. elegans and Drosophila. Dietary restriction also extends lifespan in diverse organisms, including Drosophila. Dietarily restricted flies increase the size of their digestive tract and slow down passage time of food, presumably to increase absorption of nutrients. Unpublished work has shown that flies subject to dietary restriction need only methionine to increase fecundity to the levels seen with full feeding, but that this methionine does not decrease lifespan, demonstrating that a simple competition for nutrients between somatic maintenance and reproduction cannot explain the responses of fecundity and lifespan to dietary restriction. Current data suggest that, in Drosophila at least, insulin/Igf signalling may play a substantially larger role than TOR signalling in mediating the responses of lifespan and fecundity to dietary restriction.
In his presentation, Brian Kennedy (University of Washington, Seattle, WA, USA) summarized his efforts to identify genes modulating aging in two disparate model organisms: worms and flies. The premise of these studies is that genes with conserved effects on aging in two organisms are also likely to modulate mammalian aging. Strikingly, the conserved genes that were identified converged on the TOR pathway. Mechanistic studies were performed to determine how TOR signaling is linked to aging in yeast. Findings indicate that reduced TOR activity or deletion of yeast S6 kinase (1) extends lifespan in a manner similar to dietary restriction, (2) modulates aging through control of ribosomal protein biogenesis and (3) results in enhanced translation of the stress and starvation responsive transcription factor GCN4. Since GCN4 orthologs exist in mammals, it was proposed that TOR signaling could modulate human aging through similar mechanisms. Accumulating evidence suggests that pharmacological intervention to reduce TOR or S6 kinase activity will be an effective intervention to slow aging and offset age-related disease, and. As a result, resolving the links between TOR signaling and aging will be an important question to be resolved in future studies.
In the last lecture of the meeting Christian Meyer (JP Bourgin Institute, INRA-Versailles, France) first presented a brief overview of the TOR pathway in higher plants. Plant development is continuous and highly plastic, being influenced by many environmental cues like nutrient availability and stresses. Therefore, it is very likely that the TOR signaling pathway has a strong influence on plant growth. So far, information on the roles and components of this pathway are rather scarce in plants, but it seems clear that a bona fide plant TORC1 exists. Nevertheless plants are resistant to rapamycin, probably due to mutations in residues of the FKBP12 protein which are needed to establish an inhibiting TOR-rapamycin-FKBP12 complex. Indeed expression of a yeast FKBP12 protein in Arabidopsis can restore sensitivity towards rapamycin. Conversely, there is so far no evidence for the existence of a TORC2 in plants, since no clear homologs of TORC2 protein components like Sin1, Avo2 or Rictor/Avo3 have been detected.
The disruption of both the Arabidopsis Raptor and Tor genes were found to be embryo lethal although raptor mutant embryos were arrested at an earlier stage (post-zygotic) than tor ones. A translational fusion between the Arabidopsis Tor and the GUS reporter genes allowed this group to show that the expression of the TOR protein is probably restricted to the basal growing parts of emerging young leaves and to the shoot and root meristems and conductive tissues. C. Meyer then showed that overexpression of the Tor gene in Arabidopsis leads to an increased growth of the vegetative (roots and leaves) and reproductive (inflorescence) parts and to a higher seed yield. Conversely, down-regulation of the Tor gene expression by partial or complete, ethanol-inducible, silencing produced a partial or complete growth arrest, a higher sensitivity to stress like osmotic stress, and a marked reduction in polysome abundance. The total silencing of the Tor gene resulted also in a huge starch, soluble sugar and free amino-acid accumulation and to aberrant cell division planes in the root differentiation zone. In conclusion, it seems that the plant TORC1 is central in controlling plant growth, resistance to exogenous stresses, and metabolism.
