TOR Inhibitor Research Articles

Changes of Sexual Behaviors in Rapamycin-injected Cichlid Fish Astatotilapia burtoni Males.

Cichlid fish species exhibit characteristic sexual behaviors according to not only reproductive stages but also social status. In a reproductive season, Astatotilapia burtoni males compete for females and a small number of dominant winners finally obtain the chance of spermiation. In addition to the characteristic behaviors, the dominant males have relatively bigger gonadotropin-releasing hormone 1 (GnRH1) neurons in the preoptic area (POA) of brain compared to those of subordinate males. Although the stimulatory effect of GnRH1 in vertebrate reproduction is well established, little is known about the triggering signal pathway to control GnRH1 neurons and GnRH1-mediated sexual behavior. In the present study, we evaluated the potential effect of TOR inhibitor rapamycin in relation to the cichlid male behaviors and GnRH1 neuron. After 14 h and 26 h of intraventricular injection of rapamycin, behavior patterns of chasing and courtship display did not show significant changes between rapamycin- and DMSO-injected males. Behaviors of spawning site entry increased in rapamycininjected fish at 26 h post-injection than at 14 h post-injection significantly (P<0.05). Meanwhile, there was a tendency that GnRH1 neurons’ soma size in the POA shrank by rapamycin injection, whereas the testes did not show notable changes. Taken together, these results suggest the possible role of TOR signal on GnRH1-mediated sexual behavior in cichlid dominant males, although further biological characterization of the TOR signaling pathway will be required to clarify this matter.

The effector AWR5 from the plant pathogen Ralstonia solanacearum is an inhibitor of the TOR signalling pathway.

Bacterial pathogens possess complex type III effector (T3E) repertoires that are translocated inside the host cells to cause disease. However, only a minor proportion of these effectors have been assigned a function. Here, we show that the T3E AWR5 from the phytopathogen Ralstonia solanacearum is an inhibitor of TOR, a central regulator in eukaryotes that controls the switch between cell growth and stress responses in response to nutrient availability. Heterologous expression of AWR5 in yeast caused growth inhibition and autophagy induction coupled to massive transcriptomic changes, unmistakably reminiscent of TOR inhibition by rapamycin or nitrogen starvation. Detailed genetic analysis of these phenotypes in yeast, including suppression of AWR5-induced toxicity by mutation of CDC55 and TPD3, encoding regulatory subunits of the PP2A phosphatase, indicated that AWR5 might exert its function by directly or indirectly inhibiting the TOR pathway upstream PP2A. We present evidence in planta that this T3E caused a decrease in TOR-regulated plant nitrate reductase activity and also that normal levels of TOR and the Cdc55 homologues in plants are required for R. solanacearum virulence. Our results suggest that the TOR pathway is a bona fide T3E target and further prove that yeast is a useful platform for T3E function characterisation.

Dengue Virus Inhibition of Autophagic Flux and Dependency of Viral Replication on Proteasomal Degradation of the Autophagy Receptor p62.

Autophagic flux involves formation of autophagosomes and their degradation by lysosomes. Autophagy can either promote or restrict viral replication. In the case of Dengue virus (DENV), several studies report that autophagy supports the viral replication cycle, and describe an increase of autophagic vesicles (AVs) following infection. However, it is unknown how autophagic flux is altered to result in increased AVs. To address this question and gain insight into the role of autophagy during DENV infection, we established an unbiased, image-based flow cytometry approach to quantify autophagic flux under normal growth conditions and in response to activation by nutrient deprivation or them TOR inhibitor Torin1.We found that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Early after infection, basal and activated autophagic flux was enhanced. However, during established replication, basal and Torin1-activated autophagic flux was blocked, while autophagic flux activated by nutrient deprivation was reduced, indicating a block to AV formation and reduced AV degradation capacity. During late infection AV levels increased as a result of inefficient fusion of autophagosomes with lysosomes. In addition, endolysosomal trafficking was suppressed, while lysosomal activities were increased.We further determined that DENV infection progressively reduced levels of the autophagy receptor SQSTM1/p62 via proteasomal degradation. Importantly, stable overexpression of p62 significantly suppressed DENV replication, suggesting a novel role for p62 as a viral restriction factor. Overall, our findings indicate that in the course of DENV infection, autophagy shifts from a supporting to an antiviral role, which is countered by DENV. Autophagic flux is a dynamic process starting with the formation of autophagosomes and ending with their degradation after fusion with lysosomes. Autophagy impacts the replication cycle of many viruses. However, thus far the dynamics of autophagy in case of Dengue virus (DENV) infections has not been systematically quantified. Therefore, we used high-content, imaging-based flow cytometry to quantify autophagic flux and endolysosomal trafficking in response to DENV infection. We report that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Further, lysosomal activity was increased, but endolysosomal trafficking was suppressed confirming the block of autophagic flux. Importantly, we provide evidence that p62, an autophagy receptor, restrict DENV replication and was specifically depleted in DENV-infected cells via increased proteasomal degradation. These results suggest that during DENV infection autophagy shifts from a proviral to an antiviral cellular process, which is counteracted by the virus.

A genetic screen identifies Tor as an interactor of VAPB in a Drosophila model of amyotrophic lateral sclerosis.

ABSTRACTAmyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder characterized by selective death of motor neurons. In 5–10% of the familial cases, the disease is inherited because of mutations. One such mutation, P56S, was identified in human VAPB that behaves in a dominant negative manner, sequestering wild type protein into cytoplasmic inclusions.We have conducted a reverse genetic screen to identify interactors of Drosophila VAPB. We screened 2635 genes and identified 103 interactors, of which 45 were enhancers and 58 were suppressors of VAPB function. Interestingly, the screen identified known ALS loci – TBPH, alsin2 and SOD1. Also identified were genes involved in cellular energetics and homeostasis which were used to build a gene regulatory network of VAPB modifiers. One key modifier identified was Tor, whose knockdown reversed the large bouton phenotype associated with VAP(P58S) expression in neurons. A similar reversal was seen by over-expressing Tuberous Sclerosis Complex (Tsc1,2) that negatively regulates TOR signaling as also by reduction of S6K activity. In comparison, the small bouton phenotype associated with VAP(wt) expression was reversed with Tsc1 knock down as well as S6K-CA expression. Tor therefore interacts with both VAP(wt) and VAP(P58S), but in a contrasting manner. Reversal of VAP(P58S) bouton phenotypes in larvae fed with the TOR inhibitor Rapamycin suggests upregulation of TOR signaling in response to VAP(P58S) expression.The VAPB network and further mechanistic understanding of interactions with key pathways, such as the TOR cassette, will pave the way for a better understanding of the mechanisms of onset and progression of motor neuron disease.

Abstract LB-78: Effects of rapamycin and fasting-cycles therapy on differential stress resistance and sensitization in breast cancer mouse models

Abstract Short-term starvation (STS) has been shown to be effective in both protection of normal cell and tissues and the sensitization of a variety of tumors during chemotherapy treatment. This condition of protection/sensitization of different cell types under the same condition has been named differential stress resistance (DSR). Although the effects of fasting have been mainly attributed to reduced glucose and circulating IGF1 levels, the involvement of signaling genes regulated by glucose and growth factors in these effects of fasting remain poorly understood. In this study we have administered the mTor inhibitor rapamycin in combination with STS and with the chemotherapy drug doxorubicin to shed light on the role of mTOR in the STS response and on its role in mediating DSR. In a mouse allograft model for breast cancer (4T1) we show that STS and rapamycin have an additive effect in reducing tumor progression, which confirms the possibility that STS-mediated effects on cancer cells are not due to the up-regulation of mTOR. Surprisingly, we observed that the administration of rapamycin during chemotherapy sensitizes the mice to the drug leading to an increased mortality. However, this sensitizing effect could be reversed by STS. In summary, the combination of mTor inhibition by rapamycin and STS have additive effects in retarding breast cancer tumor growth and also prevent the chemotherapy-dependent sensitization of normal cells caused by rapamycin alone. Because rapamycin is used for the treatment of certain tumors in humans, these results may have important implications for the safety of therapies using a combination of rapamycin and chemotherapy and indicate that the combination of Tor inhibitors and STS should be tested clinically. Citation Format: Stefano Di Biase, Sebastian Brandorst, Min Wei, Valter Longo. Effects of rapamycin and fasting-cycles therapy on differential stress resistance and sensitization in breast cancer mouse models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-78. doi:10.1158/1538-7445.AM2014-LB-78

Abstract 1724: Genomic mechanisms of exquisite sensitivity and acquired resistance to everolimus in a patient with anaplastic thyroid carcinoma

Abstract Understanding genetic mechanisms of sensitivity and resistance to targeted anticancer therapies may improve patient selection and rational treatment designs. One approach to increase this understanding involves the study of exceptional responders: rare patients with unexpected exquisite sensitivity or durable responses to therapy. We identified an exceptional responder on a study of the allosteric mTOR inhibitor everolimus in thyroid cancer: a 57-yr-old woman with refractory metastatic anaplastic thyroid carcinoma (ATC), a highly aggressive neoplasm with no adequate therapies and a median survival of 5 months. After beginning treatment with everolimus, the patient experienced a near complete response that lasted for 18 months, followed by progressive disease, which was then re-biopsied. To date, mechanisms of clinical resistance to mTOR inhibition have not been described. We performed whole exome sequencing (WES) of both pre-treatment and drug resistant tumor tissue to look for the underlying mechanisms of exquisite sensitivity and acquired resistance to everolimus. WES of the pre-treatment tumor revealed a somatic nonsense mutation in TSC2, a tumor suppressor gene whose inactivation is known to activate the mTOR pathway and result in sensitivity to mTOR inhibition in some cancers. WES of the drug resistant tumor additionally revealed a mutation in mTOR (mTOR-F2108L) not detected in the pre-treatment tumor. Structural modeling demonstrated that this mutation occurs in the FKBP12-rapamycin binding domain of mTOR and is predicted to prevent binding of the drug to the protein. Overexpressing mTOR-F2108L in HEK-293T cells resulted in significant resistance to rapamycin compared to cells expressing wild type (wt) mTOR. In cells expressing the mutant mTOR, rapamycin did not decrease phosphorylation of S6K1, a downstream target of mTOR, compared with cells expressing wt mTOR. Notably, cells expressing mTOR-F2108L remained sensitive to the direct TOR inhibitor torin, suggesting a therapeutic approach to overcome resistance in this patient. In summary, we add ATC to the growing list of cancers found to be exquisitely sensitive to everolimus when activating mTOR pathway mutations are present. Moreover, we present the first reported, to our knowledge, mechanism of acquired resistance to everolimus identified in patients. The fact that this occurs via a binding domain mutation that blocks allosteric mTOR inhibition suggests that followup therapy with direct TOR inhibitors may still have benefit in some patients who develop resistance to everolimus. The use of precision medicine approaches in ATC to screen for alterations in the mTOR pathway may help identify subsets of patients who would benefit from targeted therapies directed against mTOR. Moreover, the use of serial biopsies to profile patients who develop resistance to everolimus could dictate optimal followup treatment in ATC and other cancers. Citation Format: Nikhil Wagle, Brian C. Grabiner, Eliezer M. Van Allen, Ali Amin-Mansour, Scott C. Carter, Nathanael Gray, Justine A. Barletta, Scott J. Swanson, Daniel Ruan, David J. Kwiatkowski, Glenn J. Hanna, Robert I. Haddad, David Sabatini, Pasi A. Janne, Levi A. Garraway, Jochen H. Lorch. Genomic mechanisms of exquisite sensitivity and acquired resistance to everolimus in a patient with anaplastic thyroid carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1724. doi:10.1158/1538-7445.AM2014-1724

Akt regulates glutamate receptor trafficking and postsynaptic membrane elaboration at the Drosophila neuromuscular junction

The Akt family of serine-threonine kinases integrates a myriad of signals governing cell proliferation, apoptosis, glucose metabolism, and cytoskeletal organization. Akt affects neuronal morphology and function, influencing dendrite growth and the expression of ion channels. Akt is also an integral element of PI3Kinase-target of rapamycin (TOR)-Rheb signaling, a pathway that affects synapse assembly in both vertebrates and Drosophila. Our recent findings demonstrated that disruption of this pathway in Drosophila is responsible for a number of neurodevelopmental deficits that may also affect phenotypes associated with tuberous sclerosis complex, a disorder resulting from mutations compromising the TSC1/TSC2 complex, an inhibitor of TOR (Dimitroff et al., 2012). Therefore, we examined the role of Akt in the assembly and physiological function of the Drosophila neuromuscular junction (NMJ), a glutamatergic synapse that displays developmental and activity-dependent plasticity. The single Drosophila Akt family member, Akt1 selectively altered the postsynaptic targeting of one glutamate receptor subunit, GluRIIA, and was required for the expansion of a specialized postsynaptic membrane compartment, the subsynaptic reticulum (SSR). Several lines of evidence indicated that Akt1 influences SSR assembly by regulation of Gtaxin, a Drosophila t-SNARE protein (Gorczyca et al., 2007) in a manner independent of the mislocalization of GluRIIA. Our findings show that Akt1 governs two critical elements of synapse development, neurotransmitter receptor localization, and postsynaptic membrane elaboration.

TOR and S6K1 promote translation reinitiation of uORF-containing mRNAs via phosphorylation of eIF3h

Mammalian target-of-rapamycin (mTOR) triggers S6 kinase (S6K) activation to phosphorylate targets linked to translation in response to energy, nutrients, and hormones. Pathways of TOR activation in plants remain unknown. Here, we uncover the role of the phytohormone auxin in TOR signalling activation and reinitiation after upstream open reading frame (uORF) translation, which in plants is dependent on translation initiation factor eIF3h. We show that auxin triggers TOR activation followed by S6K1 phosphorylation at T449 and efficient loading of uORF-mRNAs onto polysomes in a manner sensitive to the TOR inhibitor Torin-1. Torin-1 mediates recruitment of inactive S6K1 to polysomes, while auxin triggers S6K1 dissociation and recruitment of activated TOR instead. A putative target of TOR/S6K1-eIF3h-is phosphorylated and detected in polysomes in response to auxin. In TOR-deficient plants, polysomes were prebound by inactive S6K1, and loading of uORF-mRNAs and eIF3h was impaired. Transient expression of eIF3h-S178D in plant protoplasts specifically upregulates uORF-mRNA translation. We propose that TOR functions in polysomes to maintain the active S6K1 (and thus eIF3h) phosphorylation status that is critical for translation reinitiation.

Synergism between altered cortical polarity and the PI3K/TOR pathway in the suppression of tumour growth

Loss of function of pins (partner of inscuteable) partially disrupts neuroblast (NB) polarity and asymmetric division, results in fewer and smaller NBs and inhibits Drosophila larval brain growth. Food deprivation also inhibits growth. However, we find that the combination of loss of function of pins and dietary restriction results in loss of NB asymmetry, overproliferation of Miranda-expressing cells, brain overgrowth and increased frequency of tumour growth on allograft transplantation. The same effects are observed in well-fed pins larvae that are mutant for pi3k (phosphatidylinositol 3-kinase) or exposed to the TOR inhibitor rapamycin. Thus, pathways that are sensitive to food deprivation and dependent on PI3K and TOR are essential to suppress tumour growth in Drosophila larval brains with compromised pins function. These results highlight an unexpected crosstalk whereby the normally growth-promoting, nutrient-sensing PI3K/TOR pathway suppresses tumour formation in neural stem cells with compromised cell polarity.

Rapamycin: Killing two birds with one stone

Inhibition of TOR signaling leads to extended longevity in both invertebrate and invertebrate species by modulating a number of downstream molecular pathways [1, 2]. Drugs that inhibit the TOR pathway can serve as powerful tools to translate the effects of the TOR pathway on lifespan in simpler invertebrate model systems to more complex systems like mammals. One of the most promising drugs to slow aging is rapamycin, an inhibitor of TOR, which was previously shown to extend lifespan in mice when administered late in life [3]. As aging is one of the biggest risk factors for cancer, one of the outstanding questions is whether drugs that slow aging will also slow age-related increases in cancer incidence. A recent study describes how treatment of mice initiated early in life can not only retard aging but also slow age-related increase in cancer. Work by Anisimov and colleagues examines the impact on health and lifespan of Rapamycin treated inbred female mice from 2 months of age as compared to control littermates. However, its effects on aging if administered early and intermittently have not been known. Some of the effects include a significant decline in weight gain across the lifespan, a significant increase in regularity of estrous cycle before the onset of old age and more than 20% increase in survival rate along with increase of 10% in median lifespan. Rapamycin targets TOR, a Ser/Thr kinase, the kingpin of a conserved nutrient sensing pathway and causes decline in S6 Kinase (S6K) phosphorylation at nano-molar concentrations (3). It has been earlier reported in an S6k1−/− mouse there is an increase in life span by activation of AMPK and increased resistance against many age related diseases but no effect on age-related increase in cancer incidence [4]. Hence, this study suggests that Rapamycin might have protective effects on age-related cancers through downstream effectors of TOR other than S6K. What are the possible mechanisms of decrease in age-related cancer incidence in Rapamycin treated mice? As the Rapamycin treated mice not only had a decline in number of tumor bearing mice but also showed an increase in their median lifespan, it is possible that slowing aging prevents the accumulation of age-related cancers. The authors also report an increase in chromosomal aberrations in Rapamycin treated mice as compared to control littermates. Hence protection against DNA damage is unlikely to be the cause of the protective effects of Rapamycin, however it may enhance apoptosis. Earlier higher dose of Rapamycin has been shown to be effective in killing cancerous cells through its apoptotic effects [5, 6]. Furthermore, a recent report shows a cancer cell specific apoptotic effect of high dose of Rapamycin is associated with complete dissociation of Raptor (regulatory associated protein of TOR) from mTORC1 along with decline in phosphorylation of eukaryotic binding protein-1 (4E-BP1) and inhibition of eukaryotic initiation factor 4E (eIF4E) [7]. These observations indicate the possibility that Rapamycin increases lifespan and protection against cancer by decline in phosphorylation of S6K and 4E-BP1 respectively. One possible reason for the protective effects of Rapamycin on aging in mice may the reduced mortality from cancer. Anismov et al found a reduced mortality in mice suffering from tumors [8]. However, the sample size of mice not suffering from tumors was not large to examine whether Rapamycin extends lifespan independently of its effects on cancer. Nevertheless, the study argues that treatment with Rapamycin may help prevent the onset of age-related diseases like cancer and also aging. Furthermore, given slight change in food intake, no change in length of estrous cycle and an illegible change in water intake and body temperature among treated and control groups [8] argues that Rapamycin treatment does not lead to many side effects in this study. Cancer has been linked with aging since genetic mutations accumulate with age and delayed aging has been associated with lesser incidence of cancers [9]. Activation of TOR is involved in number of aging related diseases including cancer where mutations in upstream factors like PTEN, Ras and PI3K activate TOR signaling [10]. Not all TOR mimetic have been shown to be effective against TOR dependent tumors [11], suggesting Rapamycin ingestion from early age inhibits aging that in turn provides protection against cancer. This study also provides an interesting insight for the possible use of Rapamycin in preventive medicine. Rapamycin could be given to people predisposed to cancer based on genetic and family history and may also be useful to treat cancer patients to prevent relapses and metastasis.

Role of the PI3K-TOR-S6K pathway in the onset of cell cycle elongation during Xenopus early embryogenesis

In the early embryogenesis of the frog, Xenopus laevis, cells proliferate by rapid and synchronous divisions, followed by cell cycle elongation and prolongation of the S phases, and then the appearance of the G2 and G1 phases after the midblastula transition (MBT). The beginning of cell cycle elongation was thought to depend on an increase in the nucleo-cytoplasmic (N/C) ratio in blastomeres and a decrease in cortical cytoplasmic factors necessary for cell cycle progression, although these factors are unknown. In the present study, we demonstrated that a regulatory subunit of PI3K (p85α) was localized in the cortical cytoplasm of the blastomere during the MBT. When the embryos were treated with a PI3K inhibitor, LY294002, or a TOR inhibitor, rapamycin, cell cycle elongation was initiated before the MBT. In addition, the inhibition of S6K expression by antisense morpholino oligo enhanced the initiation of cell cycle elongation. In contrast, the activation of PI3K-TOR by Rheb-S16H expression delayed the initiation of cell cycle elongation. These results indicate that a decrease in translational activity dependent on the PI3K-TOR-S6K pathway causes the initiation of cell cycle elongation at the onset of the MBT.

Rapamycin reverses hypertrophic cardiomyopathy in a mouse model of LEOPARD syndrome–associated PTPN11 mutation

LEOPARD syndrome (LS) is an autosomal dominant "RASopathy" that manifests with congenital heart disease. Nearly all cases of LS are caused by catalytically inactivating mutations in the protein tyrosine phosphatase (PTP), non-receptor type 11 (PTPN11) gene that encodes the SH2 domain-containing PTP-2 (SHP2). RASopathies typically affect components of the RAS/MAPK pathway, yet it remains unclear how PTPN11 mutations alter cellular signaling to produce LS phenotypes. We therefore generated knockin mice harboring the Ptpn11 mutation Y279C, one of the most common LS alleles. Ptpn11(Y279C/+) (LS/+) mice recapitulated the human disorder, with short stature, craniofacial dysmorphia, and morphologic, histologic, echocardiographic, and molecular evidence of hypertrophic cardiomyopathy (HCM). Heart and/or cardiomyocyte lysates from LS/+ mice showed enhanced binding of Shp2 to Irs1, decreased Shp2 catalytic activity, and abrogated agonist-evoked Erk/Mapk signaling. LS/+ mice also exhibited increased basal and agonist-induced Akt and mTor activity. The cardiac defects in LS/+ mice were completely reversed by treatment with rapamycin, an inhibitor of mTOR. Our results demonstrate that LS mutations have dominant-negative effects in vivo, identify enhanced mTOR activity as critical for causing LS-associated HCM, and suggest that TOR inhibitors be considered for treatment of HCM in LS patients.

Hot topics in aging research: protein translation and TOR signaling, 2010

In this, the fourth installment of our annual Hot Topics review on mRNA translation and aging, we have decided to expand our scope to include recent findings related to the role of TOR signaling in aging. As new data emerge, it is clear that TOR signaling acts upstream of mRNA translation, as well as a variety of other cellular processes, to modulate longevity and healthspan in evolutionarily diverse species. This Hot Topics review will cover important new findings in this area that have occurred over the past year. These include the demonstration that the TOR substrate ribosomal S6 kinase modulates longevity in mammals, the potential for TOR inhibitors as therapeutic treatments for Alzheimer's disease, and further studies emphasizing the importance of differential translation of specific mRNAs for healthy aging and enhanced longevity.

Mds3 Regulates Morphogenesis in Candida albicans through the TOR Pathway

The success of Candida albicans as a major human fungal pathogen is dependent on its ability to colonize and survive as a commensal on diverse mucosal surfaces. One trait required for survival and virulence in the host is the morphogenetic yeast-to-hypha transition. Mds3 was identified as a regulator of pH-dependent morphogenesis that functions in parallel with the classic Rim101 pH-sensing pathway. Microarray analyses revealed that mds3 Delta/Delta cells had an expression profile indicative of a hyperactive TOR pathway, including the preferential expression of genes encoding ribosomal proteins and a decreased expression of genes involved in nitrogen source utilization. The transcriptional and morphological defects of the mds3 Delta/Delta mutant were rescued by rapamycin, an inhibitor of TOR, and this rescue was lost in strains carrying the rapamycin-resistant TOR1-1 allele or an rbp1 Delta/Delta deletion. Rapamycin also rescued the transcriptional and morphological defects associated with the loss of Sit4, a TOR pathway effector, but not the loss of Rim101 or Ras1. The sit4 Delta/Delta and mds3 Delta/Delta mutants had additional phenotypic similarities, suggesting that Sit4 and Mds3 function similarly in the TOR pathway. Finally, we found that Mds3 and Sit4 coimmunoprecipitate. Thus, Mds3 is a new member of the TOR pathway that contributes to morphogenesis in C. albicans as a regulator of this key morphogenetic pathway.

MTOR favors senescence over quiescence in p53-arrested cells

The p53 tumor suppressor protein controls cell fate by inducing apoptosis or cell cycle arrest in response to cell stress [1]. Cell cycle arrest can be temporary (quiescent) or permanent (senescent) depending on p53 regulation [2, 3]. Under normal conditions, p53 binds to MDM2, which transports the protein to the cytoplasm where it undergoes rapid proteosomal degradation. A class of chemotherapeutic molecules called Nutlins inhibit p53-MDM2 interaction, and can therefore be used to control p53 activity in cancer cells [4]. In this issue of Aging, Korotchkina et al make use of nutlin-3a to dissect the mechanism by which p53 induces cellular senescence and quiescence [5]. The group demonstrates that p53-mediated senescence is irreversible in cells that maintain mTOR (mammalian target of rapamycn) signaling. However, when mTOR signaling is inhibited, activation of p53 leads to quiescence (Figure ​(Figure1).1). These findings may have broad implications because the mTOR pathway is dysregulated in many forms of cancer [6]. Figure 1. p53-induced senescence or quiescence. Establishing the mechanisms involved in cell cycle arrest and cell dormancy is critical for understanding cancer cell proliferation. Demidenko et al. have previously demonstrated that despite its ability to induce cell cycle arrest, in some cell types p53 is a suppressor, not an inducer of cellular senescence [7]. They have also shown that cells (HT-p21-9) induced into senescence using an ITPG-inducible p21 expression construct, were converted to quiescence in the presence of p53. In the same cells, nutlin-3a-induction of p53 caused reversible cell cycle arrest, and cells resumed proliferation after removal of nutlin-3a. The same group has also demonstrated that when the cell cycle is blocked, activation of mTOR is required for induction of senescence. Addition of the TOR inhibitor rapamycin converted p21-induced senescent cells back to quiescence [7]. These findings suggest that activa-tion of p53 sets in motion cell cycle arrest, after which its ability to exercise senescence is dependent on its interaction with the mTOR pathway. Senescence is achieved if p53 is incapable of disabling mTOR. Therefore, activating both mTOR and p53 in order to achieve a permanent state of cell dormancy, may prove to be a promising therapeutic strategy for treating cancer. In their current study, Korotchkina et al further explore the role of mTOR as a senescence-inducing factor. They show that nutlin-3a-induced senescent cells converted to a quiescent state when mTOR was inactivated with rapamycin (Figure ​(Figure1).1). Furthermore, the authors show that in p53-mediated quiescent cells, depletion of TSC2, a negative regulator of mTOR, results in conversion to senescence. This body of work may also offer explanations as to the role that p53 plays in aging. Others have shown that p53 function declines with age [8, 9], and mild activation of p53 may increase the lifespan of mice [10]. It will be interesting to further determine the interactions between p53 and mTOR in both models of cancer and aging.

Abstract 292: Identification of new components of the mTOR signaling pathway in pancreatic cancer cells by a genome-wide RNAi screen

Abstract The mammalian Target of Rapamycin (mTOR) is an evolutionary conserved Ser/Thr kinase involved in cell growth regulation and metabolism. Among other factors, mTOR is activated by amino acids. Although the mechanism of mTOR activation in response to growth factors and insulin is well characterized, the pathway of mTOR activation in response to amino acids is not. Previous studies have demonstrated that the latter is dominant over the former and the two pathways act independently of each other. PDAC is the most common form of pancreatic cancer and has a very dismal 5-year survival rate of 3%-5%. The need for discovery of new therapies is clear due to the low survival rates that current treatment options offer. The mTOR/ PI3K pathway is activated in pancreatic cancer and known mTOR inhibitors are currently being tested in clinical trials in pancreatic cancer patients. However, the already existing mTOR inhibitors have yet to demonstrate encouraging clinical responses. To discover druggable targets in pancreatic cancer that will be later tested in vitro and in vivo as potential therapeutics we have completed a genome-wide RNAi screen in pancreatic cancer cells in order to identify gene products whose downregulation inhibits or enhances output from the mTOR pathway. For this purpose, we used the Mia-Paca 2 cells as a representative pancreatic cancer cell line, the Dharmacon SMARTpool siRNA library and phosphorylation levels of the ribosomal S6 protein as a readout of TOR activity, since it is a well-characterized downstream target of TORC1. In initial experiments, we showed that specific RNAis against known regulators of mTORC1 activity alter baseline S6-P levels as expected with a “Z” score of 0.69. We demonstrated the success of the screen by confirming 70% of the potential positive hits from the kinome. The candidate genes found included known or previously identified components of the pathway, and also implicate many additional proteins involved in the mTOR signaling pathway. Bioinformatic analysis that integrates data obtained from this screen with those of previous studies will be presented. Immediate future studies include secondary screens with the goal of mapping where specific hits lie in the mTOR pathway and delineating the underlying mechanism of selected proteins in connection to their a) regulation of mTOR and b) their involvement in cell proliferation and apoptosis. The goal of the former is to understand the mechanism of action of selected targets in relationship to known mTOR regulators which when combined with the latter will aid in the identification of potential druggable targets. In short, we are interested in identifying and characterizing druggable targets which in combination with currently known TOR inhibitors and/ or novel ones identified through this work will sensitize pancreatic cancer cells to cell death and/ or decrease proliferation rates of pancreatic cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 292.

Effect Mechanism Of Immunosupressive Drugs

Effective immunosuppression is a key to successful organ transplantation. This study will provide an overview of different immunosuppressive agents used in solid organ transplantation. An increasing number of immunosuppressive agents are available and these agents target different steps of the immunological response to an allograft. These immunosuppressive agents are steroids, anti- proliferative agents (azathioprine and mycophenolate), calcineurin inhibitors (cyclosporine and tacrolimus), TOR inhibitors (sirolimus and everolimus), polyclonal and monoclonal antibody preparations.

Rapamycin activation of 4E-BP prevents parkinsonian dopaminergic neuron loss

Mutations in PINK1 and parkin cause autosomal recessive parkinsonism, a neurodegenerative disorder characterized by the loss of dopaminergic neurons. To highlight potential therapeutic pathways we have identified factors that genetically interact with parkin/PINK1. Here we report that overexpression of the translation inhibitor 4E-BP can suppress all pathologic phenotypes including degeneration of dopaminergic neurons in Drosophila. 4E-BP is activated in vivo by the TOR inhibitor rapamycin, which we find can potently suppress pathology in PINK1/parkin mutants. Rapamycin also ameliorates mitochondrial defects in cells from parkin-mutant patients. Recently, 4E-BP was shown to be inhibited by the most common cause of parkinsonism, dominant mutations in LRRK2. Here we further show that loss of the Drosophila LRRK2 homolog activates 4E-BP and is also able to suppress PINK1/parkin pathology. Thus, in conjunction with recent findings our results suggest that pharmacologic stimulation of 4E-BP activity may represent a viable therapeutic approach for multiple forms of parkinsonism.

Akt Phosphorylates Both Tsc1 and Tsc2 in Drosophila, but Neither Phosphorylation Is Required for Normal Animal Growth

Akt, an essential component of the insulin pathway, is a potent inducer of tissue growth. One of Akt's phosphorylation targets is Tsc2, an inhibitor of the anabolic kinase TOR. This could account for part of Akt's growth promoting activity. Although phosphorylation of Tsc2 by Akt does occur in vivo, and under certain circumstances can lead to reduced Tsc2 activity, the functional significance of this event is unclear since flies lacking Akt phosphorylation sites on Tsc2 are viable and normal in size and growth rate. Since Drosophila Tsc1, the obligate partner of Tsc2, has an Akt phosphorylation motif that is not conserved in mammals, we investigate here whether Akt redundantly phosphorylates the Tsc complex on Tsc1 and Tsc2. We provide evidence that Akt phosphorylates Tsc1 at Ser533. We show that flies lacking Akt phosphorylation sites on Tsc1 alone, or on both Tsc1 and Tsc2 concurrently, are viable and normal in size. This shows that phosphorylation of the Tsc1/2 complex by Akt is not required for Akt to activate TORC1 and to promote tissue growth in Drosophila.

Assaying autophagic activity in transgenic GFP-Lc3 and GFP-Gabarap zebrafish embryos

Autophagy mediates the bulk turnover of cytoplasmic constituents in lysosomes. During embryonic development in animals, a dramatic degradation of yolk proteins and synthesis of zygotic proteins takes place, leading to intracellular remodeling and cellular differentiation. Zebrafish represents a unique system to study autophagy due in part to its rapid embryonic development relative to other vertebrates. The technical advantages of this organism make it uniquely suited to various studies including high throughput drug screens. To study autophagy in zebrafish, we identified two zebrafish Atg8 homologs, lc3 and gabarap, and generated two transgenic zebrafish lines expressing GFP-tagged versions of the corresponding proteins. Similar to yeast Atg8 and mammalian LC3, zebrafish Lc3 undergoes post-translational modification starting at the pharyngula stage during embryonic development. We observed a high level of autophagy activity in zebrafish embryos, which can be further upregulated by the TOR inhibitor rapamycin or the calpain inhibitor calpeptin. In addition, zebrafish Gabarap accumulates within lysosomes upon autophagy induction. Thus, we established a convenient zebrafish tool to assay autophagic activity during embryogenesis in vivo.