Tài liệu Báo cáo khoa học: 1. Signal Transduction 1.1 Integration of Metabolism and Survival pdf

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1. Signal Transduction1.1 Integration of Metabolism and SurvivalS1.1-1Regulation of metabolism by estrogensignalingJ. Gustafsson, R. Rodrigo and W. WarnerDepartment of BioSciences and Nutrition, Novum, KarolinskaInstitutet, Stockholm, Sweden.E-mail: jan-ake.gustafsson@mednut.ki.seEstrogen is known to influence glucose homeostasis but the roleof estrogen receptors in muscle glucose metabolism is unknown.Therefore, we investigated the expression of the two estrogenreceptors, ERa and ERb and their influence on regulation ofGLUT4, and its associated structural protein, caveolin-1, inmouse muscle. ERa and ERb are co-expressed in the nuclei ofmost muscle cells and their levels were not affected by absence ofestradiol (in aromatase knockout, ArKO, mice). GLUT4 expres-sion on the muscle cell membrane was not affected by loss ofERb but was extremely reduced in ERa–/–mice and elevated inArKO mice. Upon treatment of ArKO mice with the ERb agon-ist, DPN, GLUT4 expression was reduced. Caveolin-1 expressionwas higher in ArKO mice and lower in ERb–/–and ERa–/–micethan in WT littermates. GLUT4 and caveolin-1 were colocalizedin WT and ArKO mice, but not in ERb–/–and ERa–/–mice.Thus, ERa is necessary for GLUT4 expression while ERb has asuppressive role. Both ERb and ERa are necessary for optimalcaveolin-1 expression. Taken together, these results indicate thatreduction in GLUT4 is a plausible explanation for the insulinresistance observed in ERa)/)mice and that colocalization ofcaveolin-1 and GLUT4 is not an absolute requirement for muscleglucose metabolism.S1.1-2Nutritional sensing in the MTOR/S6K1pathway in the development of obesity anddiabetesG. ThomasGenome Science, Genome Reserach Institute, Cincinnati, OH,USA. E-mail: thomasg4@uc.eduEarlier we showed that nutrient overload induces S6K1 activa-tion, which suppresses insulin-induced class 1 PI3K signalingleading to insulin resistance. Counter to the prevailing view, ourrecent studies show that the nutrient, e.g. amino acid (AA), inputto S6K1 is not mediated by the tumor suppressor TSC1/TSC2 orits target the oncogene Rheb. In the absence of TSC1/2, we findS6K1 is activated and refractile to insulin, but still regulated byAAs. However this is not the case for Rheb, as siRNA knock-down of Rheb protein levels blocks both the insulin and AAinput to S6K1. Nonetheless, withdrawal of AAs has no effect onRheb-GTP levels, but triggers S6K1 inactivation, indicatingRheb-GTP is necessary but not sufficient for AA induced S6K1activation. This suggested the AA input to S6K1 is on a parallelpathway to the TSC1/2-Rheb axis. That wortmannin, a class 1PI3K inhibitor, blocks AA-induced S6K1 activation but AAs donot induce PKB activation, suggested a novel wortmannin sensi-tive signaling component mediated the AA input to S6K1, whichwe identified as class 3 PI3K, hVps34. In brief, ectopic expressionof hVps34 drove S6K1 activation, an effect blocked by hVps34siRNAs. Moreover, AAs increase hVps34 activity and the pro-duction of PI3P, which serves to recruit FYVE or Px containingproteins to endosomes to build signaling platforms. Consistentwith this, S6K1 activation is attenuated by ectopic expression ofa dominant interfering cDNA containing two FYVE domains.S1.1-3Regulation and function of the WNK1 andWNK4 protein kinasesD. AlessiMRC Protein Phosphorylation Unit, University of Dundee,Dundee, UK. E-mail: d.r.alessi@dundee.ac.ukThe WNK family of protein kinases were originally identified asenzymes that lack a conserved Lys residue normally found insubdomain II of the catalytic domain. Subsequent studies, identi-fied mutations in the genes encoding WNK1 and WNK4, in fam-ilies with an inherited hypertension and hyperkalaemia disorder,called PHAII/Gordon1s syndrome. WNK isoforms are large pro-tein kinases (WNK1-2382 residues, WNK4-1243 residues), inwhich the catalytic domain is located at the N-terminus. Apartfrom two putative coiled-coil domains, the remainder of theWNK polypeptides possess no obvious structural features. Muta-tions in the WNK1 gene found in PHAII subjects, are deletionsin intron-1, which elevate the expression of the WNK1 protein,indicating that hypertension could result from increased expres-sion of WNK1. Consistent with this notion, mice lacking oneallele of WNK1, had lower blood pressure. Thus far, the muta-tions in the WNK4 gene found in PHAII subjects, lie distal toboth of the putative coiled-coil domains. Little is known aboutthe molecular mechanism by which WNK isoforms regulate cel-lular processes. In my talk I will present our recent results thatindicate that the WNK protein kinases are activated by osmoticstress and phosphorylate and activate protein kinases of theSTE20 family, termed STE20/SPS1-related Proline-Alanine-richKinase (SPAK) and the Oxidative Stress Response kinase-1(OSR1).Abstracts41.2 Integration of Defence and SurvivalS1.2-1Immuno-receptors recognition of antigens: theT-cells caseD Gakamsky and I. PechtImmunology, Weizmann Institute of Science, Rehovot, Israel.E-mail: israel.pecht@weizmann.ac.ilResolution of the interaction mechanism between the T-cell recep-tor (TCR) and its ligands, MHC peptide (MHCp) complexes, isstill a central problem in immunology. These are characterized rel-atively high specificity and modest affinities. 3-D structures ofTCR-MHCp complexes revealed marked changes are observed,primarily in the TCR, upon ligand binding the. Moreover, bothtime course and affinity measurements of TCR-MHCp interactionsobtained using the Surface Plasmon Resonance (SPR) methodestablished the 0.1–100 mM affinity range for the interactions ofthe different TCR-MHCp couples examined. These were inter-preted to be a result of relatively slow association (103–105/M/s)and fast complex dissociation (0.1–100/s) rate constants. However,practically all values for the association rate constants were calcu-lated by assuming the operation of a reversible, single step reactionmechanism. This conflicts with the crystallographic evidence forconformational changes taking place in the TCR upon interactionwith its ligands, and calls for considering a more complex mechan-ism for MHCp-TCR interaction. Indeed, an in-depth analysis ofkinetic data obtained by SPR as well as an independent, FRETbased study of TCR-MHCp interactions shows that these fit welltwo different, though related mechanisms, both involving conform-ational transitions coupled to the binding process.S1.2-2Amplification and control of B cell antigenreceptor signalingM. Reth and Y. KulathuInstitute for Biology III and Max-Planck-Institute forImmunobiology, Freiburg, Germany.E-mail: reth@immunbio.mpg.deSignal transduction from the B cell antigen receptor (BCR)involves not only the activation of BCR-proximal protein tyro-sine kinases (PTK) like Syk and Lyn, but also the inhibition ofprotein tyrosine phosphatase (PTP) like SHP-1 through the oxi-dation of its catalytic active cysteine. For signaling it is thereforeimportant that the BCR is localized to areas of high productionof oxidants and low PTP activity. Under these condition Syk isreleased from auto-inhibition by binding to the BCR and canamplify the BCR signal through ITAM phosphorylation. Theactivation of Syk and its release from auto-inhibition involves atthe same time a conformational change and its translocation tothe plasma membrane via binding of its tandem SH2 domain tophosphorylated ITAMs. To uncouple these two important eventsin Syk activation, we have targeted Syk permanently to the mem-brane and tested a panel of Syk mutants for their function. Thesedata show that several parts of the Syk molecule are involved inthe auto-inhibition control. Once resuming an open conforma-tion, Syk can recruit other signalling molecules thus facilitatingtheir phosphorylation and the propagation of the BCR signal.These studies, which are conducted in a novel signalling pathwayreconstitution system with S2 Schneider cells, give a moredetailed inside into the mechanistic aspect of BCR signaling.S1.2-3Temporal regulation of cytolytic T cellprogrammingG. Verdeil, J. Chaix, A. Guimezanes, N. Auphan-Anezin andA. Schmitt-VerhulstCentre d’Immunologie de Marseille-Luminy, CNRS-INSERM-Univ. de la Me´diterrane´e, Marseille, France.E-mail: verhulst@ciml.univ-mrs.frNaı¨ve CD8 T cells may develop into cytolytic effectors followingengagement of their clonally expressed antigen receptor (TCR),thus committing the T cells to a program that includes clonalexpansion, differentiation, and long-term survival. Incompletedifferentiation has been reported, however, in particular for weaktumor antigens. To understand the molecular basis for the plasti-city in CD8 T cell differentiation, we analyzed the response ofmonoclonal naı¨ve CD8 T cells to antigenic ligands of differentaffinity. A transient versus stable pattern of gene expression wasinduced by weak versus strong TCR agonists, respectively. Forthese transcripts, including those of the IL-2 receptor (IL-2R),cytolytic effector molecules (granzyme B, perforin) and TNF-Rfamily members (GITR, OX40, 4-1BB), expression was sustainedupon addition of IL-2, an effect mimicked by expression of anactive form of STAT5. Epigenetic modifications of histones asso-ciated with relevant genes were delayed in response to weakagonists. Addition of IL-2 failed to shorten this delay, butinduced STAT5 binding to granzyme B and 4-1BB promoters. Adelayed and weak ERK1/2 activation in response to weak TCRagonists was correlated with a delayed onset of proliferation. Wethus identified the TCR, IL-2R and members of the TNF-R fam-ily, as sequential contributors to the proliferation, differentiationand survival of effector CD8 T cells, involving ERK, STAT5 andNF-kB signaling pathways, respectively.1.3 Rhythmic Signals: The Setting of Biological TimeS1.3-1An ultradian clock in yeast: metronome forintracellular coherenceD. Lloyd1and D. Murray21Microbiology Group (Biosi 1) Cardiff University, Cardiff, Wales,UK,2The Systems Biology Instititue, Shinanomachi ResearchPark, Keio University School of Medicine, Shinjuku-ku, Tokyo,Japan. E-mail: lloydd@cf.ac.ukSelf-synchrony of a densely-populated culture of Saccharomycescerevisiae in aerobic continuous culture under controlled condi-tions of stirring, temperature and pH reveals an ultradian (with aperiod of about 40\,min) clock. The high amplitude respiratoryoscillation is robust in that it can be maintained over extendedtimes(up to months) and its period is compensated for tempera-ture and nutrtional changes. Extensive studies of metabolism andmicromolecular synthesis indicate that an oscillatory alternationbetween oxidative and reductive states underlies the observedoutputs (mitochondrial respiratory activity, nicotinamide indica-ated by nucleotides reduced glutathione content, mitochondrialinner membrane potential and levels of transcripts). Ultra-structural changes also occur during redox state transitions;Abstracts5mitochondrial matrix volume indicates energization states. Wesuggest that this timekeeping mechanism serves as the universalcoordinating time-base upon which all intracellular reactions,pathways, processes and events are dependent for synchronouscoordination and coherence. Circadian control systems thatmatch internal activities to environmental changes may use ultra-dian rhythyms as their bases. The observables of the culture pro-vide us with a unique insight into the temporal organization ofthe living state in every cell and at all times.Lloyd D. and Murray D. B. Ultradian metronome: timekeeperfor orchestration of cellular coherence. TIBS 30: 373–377.S1.3-2The mammalian circadian timing system: fromcyclic transcription to rhythmic physiologyB. Kornmann, F. Gachon, H. Reinke, J. Ripperger,G. LeMartelot and U. SchiblerDepartment of Molecular Biology and NCCR Frontiers inGenetics, University of Geneva, Geneva, Switzerland.E-mail: ueli.schibler@molbio.unige.chCircadian pacemakers were originally believed to exist only in afew specialized cell types, such as SCN neurons. However, inrecent years, this view has been challenged by the discovery thatself-sustained and cell-autonomous circadian clocks are operativein most peripheral organs. We are studying how such peripheraloscillators can drive overt cycles in physiology. The PAR basicleucine zipper (PAR bZip) proteins DBP, HLF, and TEF serveas clock output regulators. Their expression is governeddirectly by cellular circadian oscillators. In turn, the rhythmicaccumulation of these proteins results in daily cycles of targetgene expression. Transcriptome profiling studies with PAR bZipdeficient knockout mice revealed that DBP, HLF, and TEF regu-late primarily the transcription of genes involved in hepatic,renal, and intestinal detoxification and genes of the innate andadaptive immune system. REV-ERBalpha, a nuclear orphanreceptor acts as a strong transcriptional repressor when bound toRORE elements. It generates circadian expression cycles ofBMAL1 and CLOCK, the two positive limb members of themolecular oscillators, by periodically repressing the transcriptionof these genes. In addition, REV-ERBalpha governs the circadianexpression of several enzymes and regulators of lipid metabolism.Our genetic Rev-Erbalpha loss-of-function and gain-of-functionstudies suggest that circadian transcription in the periphery canbe governed by cell-autonomous and systemic cues.S1.3-3Central and peripheral clocks in ontogenyA. Sumova, Z. Bendova, M. Sladek, R. El-Hennamy,K. Laurinova, Z. Jindrakova and H. IllnerovaDepartment of Neurohumoral Regulations, Institute of Physiology,Academy of Sciences of the Czech Republic.E-mail: sumova@biomed.cas.czIn mammals, endogenous rhythmicity of the principal circadianclock located within the suprachiasmatic nucleus (SCN) isentrained predominantly by a light–dark cycle through pathwaysemanating from retinal photoreceptors. Via separate pathways,non-photic cues may also reset the clock. The SCN clock entrainscircadian clocks in numerous peripheral tissues and controlsrhythms of various body functions. During ontogeny, themolecular mechanism responsible for generation of the circadianrhythmicity develops gradually from prenatal to postnatal periodboth in the SCN and in peripheral organs. At the beginning, thematernal SCN sets the phase of the developing fetal and earlypostnatal clocks. In altricial rodents, the external light–dark cyclestarts to reset the SCN clock only after the first postnatal week.However, full adjustment to the day-length, i.e. to the photope-riod, is accomplished only around the time of weaning. Appar-ently, maternal and photic resetting of the developing circadianclocks employ different and non converging signaling pathways.While the photic entrainment pathways have already beenroughly recognized, maternal entrainment pathways are still onlysuggested. The data will summarize the current knowledge onmechanism of setting the time of the developing SCN and periph-eral clocks. It appears that signaling from mothers and from theoutside world to the newborn circadian clocks might be verycomplex.1.4 NF-jB Pathway in Normal Physiology and DiseaseS1.4-1The IKK complex-linking inflammation tocancerM. KarinDepartment of Pharmacology, University of California, San DiegoLa Jolla, CA, USA. E-mail: karinoffice@ucsd.eduA link between inflammation and cancer has been suspected forover two millennia, but its molecular nature remained ill defined.It has also been observed that certain bacterial (for instance Heli-cobacter pylori) and viral (for instance HBV and HCV) patho-gens are major risk factors for certain types of cancer, mostnotably gastric and liver cancers. We have postulated that tran-scription factor NF-jB may be at the center of this nexus, asNF-jB is activated in response to infection and inflammationand in turn upregulates expression of anti-apoptotic and growthpromoting genes. As there are several NF-jB transcription fac-tors, we decided to inactivate the critical catalytic subunit of theIjB kinase (IKK) complex, IKKb, as a way to inhibit activationof most NF-jB forms. While inflammation is a major factor thatcontributes to the development and progression of CAC andother inflammation-linked cancers and is estimated to be involvedin up to 20% of all human cancers, we asked whether inflamma-tion driven by NF-jB has an important role in other forms ofcancer where chronic inflammation or infection do not precedetumor development. To that end, we used a model of chemically-induced hepatocellular carcinoma (HCC) based on exposure ofmice to a complete and potent carcinogen – diethyl nitrosamine(DEN). Heretofore, DEN administration, although resulting inpronounced cytotoxicity, was not found to trigger an inflamma-tion response.S1.4-2NF-jB signalling in muscle regenerationN. Rosenthal, F. Mourkioti, E. Lara-Pezzi and M. PasparakisMouse Biology Unit, EMBL-Monterotondo, Rome, Italy.E-mail: rosenthal@embl.itThe adult mammalian body does retain the robust repair capa-city of the embryo into adulthood and gradually loses its regener-ative potential. Our approach has been to intervene in themechanisms at work in the mammalian response to damage orAbstracts6disease by reducing the impediments to effective regeneration ofskeletal muscle. In one intervention, transgenic supplementationof a locally acting Insulin-like Growth Factor 1 isoform (mIGF-1) promotes efficient tissue repair of damaged skeletal and car-diac muscle without scar formation, and prevents muscle atrophyin heart failure. In a second intervention, repression of the NFjBinflammatory pathway by mIGF-1 in damaged muscle hasprompted studies in which mice lacking functional NFjB signal-ling specifically in skeletal muscle exhibit increased muscle regen-erative capacity. In a second intervention, supplementalexpression of a naturally occurring, active calcineurin isoform(CnAb1), which is normally induced transiently during skeletalmuscle regeneration and in response to mIGF-1 in atrophyingmuscle, enhances repair and reduced fibrosis in response to dam-age, and activates repressors of the NFjB pathway. Takentogether, these observations support the feasibility of recapturingembryonic regenerative capacity by modulating key signallingpathways in the adult to restore injured or degenerating tissues.S1.4-3The biology of NF-jB interactions withchromatinG. NatoliDepartment of Experimental Oncology, European Institute ofOncology, Milan, Italy. E-mail: gioacchino.natoli@ifom-ieo-cam-pus.itNF-jB is a family of rapidly activated transcription factors con-served in multi-cellular eukaryotes, whose main role is to controlinducible transcription of most inflammatory genes, as well asgenes involved in multiple steps of cancer development and pro-gression. Overall, we know far less about the mechanisms regula-ting NF-jB activity in the nucleus than about signalingmechanisms responsible for its activation.Understanding NF-jB-regulated transcription is a crucial task todecode the logic of physiological and abnormal inflammatoryresponses and it should provide general paradigms about controlof inducible transcription in higher eukaryotes. How are NF-jB:DNA transactions regulated? How are NF-jB-dependentgenes with different functions expressed with kinetics that suittheir function? How different microbes and inflammatory stimulican induce transcriptional outputs that are plastically adapted toeach input? Transcriptional specificity may represent a partic-ularly relevant task in the context of the anti-microbial response,since rapid and successful elimination of any pathogen willrequire the induction of a suitable transcriptional program. Inmy talk I will review some recent advancements in this field andtheir implications.1.5 Signaling and Cancer: Nuclear Receptor ConnectionS1.5-1Androgen action and prostate carcinogenesisF. Saatc¸ iog˘luDivision of Cellular and Molecular Biology, Department ofBiology, University of Oslo, Oslo, NorwayAndrogens have critical roles in the development and mainte-nance of the male reproductive system and important for pro-gression of prostate cancer. Using a simple screening protocol,we have cloned androgen responsive genes that are also enrichedto prostate for expression. The characterization of three of thesegenes and the proteins that they encode will be presented: Kallik-rein 4 (KLK4), six transmembrane protein of prostate 1(STAMP1), and STAMP2. KLK4 belongs to the KLK family,which includes Prostate Specific Antigen (PSA, or KLK3), asecreted protein that is widely used as a diagnostic marker forprostate cancer. KLK4 is regulated by androgens and is highlyspecific to prostate for expression. Interestingly, KLK4 is the firstmember of the KLK family that is intracellularly localized.KLK4 is predominantly expressed in the basal cells of the normalprostate gland and overexpressed in prostate cancer. STAMP1and STAMP2 are six transmembrane proteins. WhereasSTAMP1 is largely specific to prostate for expression, STAMP2has a wider tissue distribution and its expression is exquisitelyregulated by androgens. Live cell imaging using STAMPs fusedwith green flourescent protein (GFP) show that STAMPs shuttlebetween the trans-Golgi network (TGN) and the plasma mem-brane suggesting that they may be involved in the secretory/endo-cytic pathways. STAMP1 is expressed exclusively in the epithelialcells of the prostate and its expression is significantly increased inprostate tumours compared with normal glands, whereasSTAMP2 is highly overexpressed in a subset of the prostate ade-nocarcinoma specimens compared with normal prostate epithelialcells. Ectopic expression studies indicate that these genes may beinvolved in proliferation of prostate cancer cells. Taken together,these data suggest that KLK4, STAMP1, and STAMP2 contri-bute to the normal biology of the prostate cell, as well as pros-tate cancer progression. In addition to these data, the molecularmechanism of antiandrogen action will be presented, determinedby live cell imaging of GFP-tagged androgen receptor (AR) inter-acting with its target sites.S1.5-2Transcription factor mobility and promoterprogressionG. L. Hager, R. L. Schiltz, M. Wiench, T. Johnson, S. John,A. Nagaich and Y. QiuLaboratory of Receptor Biology & Gene Expression, NCI, NIH,Bethesda, MD, USA. E-mail: hagerg@exchange.nih.govThe classical view of nuclear receptor action postulates the staticbinding of liganded receptors to the promoter. We discovered,however, that nuclear receptors interact dynamically with regula-tory elements in living cells, and have proposed the hit and runhypothesis for receptor function. We have also observed thatsteroid receptor responsive promoters move through a complexseries of activity states, a phenomenon we term promoter pro-gression. Genome-wide profiling of glucocorticoid receptor (GR)regulated loci reveals several classes of response, including genesthat are transiently activated and genes that are transientlyrepressed. Thus receptor action either leads to a series of eventsAbstracts7programmed into each promoter, or the receptor and/or associ-ated factors are subject to a time dependent modification of theiractivity states. We have discovered that HDAC1 is a coactivatorfor GR induction of MMTV. A sub-fraction of HDAC1 presentin a complex with GR becomes acetylated upon hormone treat-ment. This acetylated form of HDAC1 appears in the GR com-plex when MMTV transcription is inhibited, indicating thatacetylation of HDAC1 plays a repressive role on MMTV tran-scription. These findings suggest that a critical component of theGR responsive transcriptional apparatus requires deacetylationby HDAC1 for activity, and inactivation of the enzyme duringthe induction cycle inhibits this process, thus shutting down thepromoter after a period of transient activation.S1.5-3Analysis of steroid hormone receptor functionby gene targetingG. Schu¨tzMolecular Biology of the Cell I, German Cancer Research Center,Heidelberg, Germany. E-mail: g.schuetz@dkfz.deGermline and somatic gene targeting of genes for steroid hor-mone receptors allows the characterization of their functions aswell as their molecular modes of action. For the glucocorticoidreceptor (GR) multiple modes of action have been identified. Thereceptor activates expression of genes by binding as a dimer toglucocorticoid response elements (GRE) as well as by interactionas a coactivator with DNA-bound Stat5. The receptor is able torepress expression of genes by protein–protein interaction and bybinding to negative GREs. Cre/loxP-mediated generation ofsomatic mutants of the mineralocorticoid receptor (MR) circum-vents the early lethality observed after germline inactivation.Inactivation of MR in the forebrain leads to impaired hippocam-pal-dependent learning, but the limbic MR is dispensable for themaintenance of basal hypothalamic–pituitary–adrenal axis activ-ity. The mechanisms underlying the critical actions of estrogen inthe secretion of the gonadotropin-releasing hormone (GnRH) areunknown. A neuron-specific ERa mutation in the forebrain leadsto infertility and loss of the positive feedback effects of estrogenupon GnRH neurons. As GnRH neurons do not express ERal-pha, these results indicate that ERalpha-expressing neuronal aff-erents to GnRH neurons are critical for the preovulatory GnRH/LH surge. These genetic approaches not only reveal novel neuralfunctions of these regulatory molecules in gene expression, butalso unprecedented modes of their activity.1.6 Cell Surface Receptors and Downstream TargetsS1.6-1Small GTPase signalling pathways in tumourbiologyC. J. MarshallOncogene Team, Cell and Molecular Biology, Institute of CancerResearch, London, UK. E-mail: chris.marshall@icr.ac.ukSmall GTPases of the Ras, Rho and Ral families play importantroles in tumour biology. Genetic alterations to small GTPasesunderscore this role. Ras is mutated in some tumours while wildtype RhoA and RhoC are over-expressed particularly in moreaggressive tumours. As well as their individual roles it is emer-ging that there are significant interactions between small GTPasesignalling pathways. Interesting examples of such interactions areemerging through studies on invasion/cell motility. A number ofthe transcriptional targets of ERK-MAP kinase signalling down-stream of oncogenic Ras are involved in cell movement andadhesion. Others act through regulating the activation state ofRho-family signalling pathways, for example by down-regulationof ROCK or Rho activation and up-regulating Rac activation.S1.6-2Signal transduction via receptors for PDGFand TGF-beta – possible targets for tumortherapyC H. HeldinLudwig Institute for Cancer Research, Uppsala University,Uppsala, Sweden. E-mail: c-h.heldin@licr.uu.sePlatelet-derived growth factor (PDGF) and transforming growthfactor-beta (TGF-beta) affect cell growth, survival and migration,and have important functions during the embryonal develop-ment. PDGF isoforms exert their cellular effects via two structur-ally similar tyrosine kinase receptors. Since PDGF promotes cellgrowth and survival, overactivity of the PDGF signaling pathwayis associated with diesease, e.g. malignacies. We have exploredthe use of PDGF antagonists in tumor treatment, and found effi-cient inhibition of tumor growth in animal models of tumors dri-ven by autocrine PDGF production. In addition, we haveobserved that inhibition of paracrine PDGF stimulation of stro-mal fibroblasts and vessel pericytes lowers tumor interstitial fluidpressure and tumor angiogenesis. TGF-beta has a more compli-cated role in cancer; initially TGF-beta is a tumor suppressorthrough its ability to inhibit growth and to promote apoptosis oftumor cells. At later stages, when tumor cells become insensitiveto the cytostatic effects of TGF-beta, TGF-beta has tumor pro-moter effects through stimulation of epithelial-to-mesenchymaltransition of tumor cells, stimulation of angiogenesis andsuppression of the immune system. We are currently delineatingthe signaling pathways involved in the various cellular effects ofTGF-beta, and exploring the possible use of TGF-beta antago-nists in tumor treatment.S1.6-3New functions of focal adhesion kinase: amajor Src effector involved in cancerM Frame and B SerrelsBeatson Institute, Glasgow, UK.E-mail: m.frame@beatson.gla.ac.ukFAK expression is elevated, often at the level of gene dosage/amplification, in epithelial cancer cells. We reported the firstskin-specific conditional knockout of FAK using Cre-lox technol-ogy. Skin carcinogenesis experiments revealed that FAK defici-ency leads to reduced incidence of papillomas and inhibition ofprogression to carcinomas. Mechanistically, loss of FAK is asso-ciated with increased apoptosis in both keratinocytes in vitro andin the skin in vivo, particularly in the hair follicle bulge regionwhere the stem cell targets for tumorigenesis reside. We haverecently expressed putative gain-of-function and loss-of-functionmutants of FAK as skin-targeted transgenes, and have found thatelevated FAK expression can cause accelerated carcinogenesisAbstracts8and progression to carcinoma. In addition to our in vivo experi-ments, in vitro experiments have identified a new function of theFAK FERM domain in regulating actin assembly via the Arp2/3complex. Specifically, the FERM domain binds and recruits theArp2/3 complex to peripheral adhesion sites. Critically, Arp2/3 issubsequently released from complex with FAK by integrin-induced auto-phosphorylation of FAK-tyrosine-397, which liesadjacent to the FAK FERM domain. Moreover, release ofArp2/3 is associated with recruitment of actin into filaments inthe vicinity of focal adhesions. These data suggest new ways inwhich integrin and Src signalling to FAK may contribute to thecancer phenotype.1.7 Signaling Through Ion ChannelsS1.7-1Voltage-gated sodium channel upregulation asan accelerating factor in cancer cell behaviourM.B.A. DjamgozDivison of Cell & Molecular Biology, Imperial College London,London, UK. E-mail: m.djamgoz@imperial.ac.ukElectrophysiological recordings from contrasting strongly versusweakly metastatic human prostate cancer (PCa) and breast can-cer (BCa) cells showed that the former specifically expressedfunctional voltage-gated sodium channels (VGSCs). At mRNAlevel, the upregulation was greater than 1000-fold. Similar upre-gulation of VGSC mRNA and protein was detected in humanbiopsies. Blocking VGSC activity with the highly specific neuro-toxin, tetrodotoxin (TTX), suppressed the cells’ metastatic behav-iours (MCBs) in vitro. Thus, lateral motility (includinggalvanotaxis), transverse migration, endocytic membrane activity,adhesion and Matrigel invasiveness were modulated by 50%,consistent with the notion that VGSC activity would potentiatemetastasis. Semi-quantitative PCR measurements also revealedthat Nav1.7 and Nav1.5 were the culprit VGSCs in PCa andBCa, respectively. Importantly, in both cancers, the VGSC wasexpressed in its neonatal splice form. In the case of BCa, the neo-natal splice variant differed from the adult in a string of sevenamino acids of unique sequence and a polyclonal antibody couldbe raised to target the VGSC protein. Blocking VGSC activity inthe human BCa MDA-MB-231 cell line with either the antibodyor siRNA, again, suppressed MCB by 50% and eliminated theTTX sensitivity, i.e. the potentiating effect of VGSC. It is conclu-ded that VGSC expression/activity is a viable novel target forclinical management of metastatic disease.S1.7-2The role of EAG, a potassium channel, incancerW. Stu¨hmerMBNS, Max-Planck-Institute of Experimental Medicine, Go¨ttin-gen, Germany. E-mail: wstuehm@gwdg.deIon channels are increasingly being linked to cancer and tumourprogression. Here we describe a voltage-gated, potassium select-ive channel (EAG or ether-a-go-go) with novel electrophysiologi-cal properties, whose normal physiological function is yetunknown but which acts as an oncogene if expressed ectopicaly.One of the most characteristic properties of the channel, its ionicselectivity, is modulated during cell-cycle transitions. Strikingly,the expression of the human EAG is restricted to brain, but it isalso present in several tumour-derived cell lines. While normaltissue is not stained with EAG antibodies, over 75% of tumourstested showed positive staining. Experiments under in vitro condi-tions have demonstrated decreased proliferation of EAG1-expres-sing cells by inhibition of this channel. This inhibition of EAG1is accomplished using RNA interference, functional anti-EAG1antibodies, or (unspecific) EAG1 channel blockers. We have usedthe eXplore Optix system to visualize the distribution of EAG inan in vivo mice tumour model. For this, a specific recombinantFab fragment against EAG was labelled either with DsRed orCy5.5. These IR-fluorescent antibody constructs were injectedinto immuno-suppressed mice carrying previously grafted MDA-MB-435 S human mammary carcinoma cells. This enabled us tofollow tumour localization as well as tumour progression. Weconclude that EAG is a widely distributed tumour marker withdiagnostic and therapeutic potential.S1.7-3Cyclic nucleotide-gated ion channelsA. Menini, A. Boccaccio and S. PifferiNeurobiology Sector, International School for Advanced Studies,SISSA, Trieste, Italy. E-mail: menini@sissa.itIon channels activated by the binding of cyclic nucleotides arepresent in several types of cells. Their main physiological roleconsists in translating changes in the concentration of cAMP orcGMP into a variation of the membrane potential. Cyclic nuc-leotide-gated (CNG) channels were first discovered in the outersegment of retinal rods and shortly after in the cilia of olfactorysensory neurons. In these sensory systems CNG channels medi-ate sensory transduction by conducting cationic currents carriedprimarily by sodium and calcium ions. In olfaction the bindingof odorants to olfactory receptors in the cilia causes, via G pro-tein activation of adenylyl cyclase, the increase of intracellularcAMP, which directly opens CNG channels. The increase inintracellular calcium entering through CNG channels has bothinhibitory and excitatory feedback effects. The opening ofCa-activated Cl channels increases the transduction currentbecause olfactory sensory neurons maintain a high intracellularCl concentration, whereas calcium in combination with calmodu-lin exerts a negative feedback on CNG channels. By using thewhole-cell voltage-clamp technique to record the current, incombination with photolysis of caged cAMP or 8-Br-cAMP, weshowed that the Ca feedback on CNG channels is the mainmolecular mechanism responsible for fast adaptation in olfactorysensory neurons.Abstracts9 . 1. Signal Transduction 1. 1 Integration of Metabolism and Survival S1 .1- 1Regulation of metabolism by estrogensignalingJ. Gustafsson, R. Rodrigo and. kinase -1 (OSR1).Abstracts4 1. 2 Integration of Defence and Survival S1.2 -1 Immuno-receptors recognition of antigens: theT-cells caseD Gakamsky and I.
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