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1、如何阅读文献,在肿瘤学方面有哪些好的杂志如何快速阅读文献以获取知识论文的主要结构,肿瘤的分子信号转导,+genomic instability,from Hanahan and Weinberg 2000,Signal Transduction and Cancer,Lecture I:Growth Factors and ReceptorsOutline:What is Signal Transduction?What are Growth Factors?How do they contribute to normal ST?How is this ST deregulated in Ca
2、ncer?,Lecture I:Growth Factors and ReceptorsWhat is Signal Transduction?Signal Transduction is the process by which a cell converts an extracellular signal into a response.Involved in:Cell-cell communicationCells response to environmentIntracellular homeostatsis-internal communication,Generic Signal
3、ling Pathway,SignalReceptor(sensor)Transduction CascadeTargetsResponse,Altered Metabolism,MetabolicEnzyme,Gene Regulator,Cytoskeletal Protein,Altered Gene Expression,Altered Cell Shape or Motility,Adapted from Molecular Biology of the Cell,(2002),4th edition,Alberts et al.,Components of Signalling,W
4、hat can be the Signal?External message to the cell,Peptides/Proteins-Growth FactorsAmino acid derivatives-epinephrine,histamineOther small biomolecules-ATPSteroids,prostaglandinsGases-Nitric Oxide(NO)PhotonsDamaged DNAOdorants,tastants,Signal=LIGANDLigand-A molecule that binds to a specific site on
5、another molecule,usually a protein,ie receptor,Components of Signalling,What are Receptors?Sensors,what the signal/ligand binds to initiate ST,Cell surface Intracellular,Hydrophillic Ligand,Cell-Surface Receptor,Plasma membrane,Hydrophobic Ligand,Carrier Protein,IntracellularReceptor,Nucleus,Adapted
6、 from Molecular Biology of the Cell,(2002),4th edition,Alberts et al.,Cell Surface Receptor Types:Ligand-gated ion channel,Cell Surface Receptor Types:2)G-Protein Coupled Receptor,Cell Surface Receptor Types:3)Enzyme-linked Receptor eg Growth Factor Receptors,Growth Factors,Ligands which bind enzyme
7、 linked receptorsSignal diverse cellular responses including:ProliferationDifferentiationGrowthSurvival AngiogenesisCan signal to multiple cell types or be specific,Growth Factor Receptors,Most growth factors bind Receptor Tyrosine Kinases,Growth Factor Receptor Activation I,RTK,RS/TK,Growth Factor
8、Receptor Activation II,Growth signal autonomy,Insensitivity to anti-growth signals,Resistance to apoptosis:Uncouple cells growth program from signals in the environment.Growth factors in normal cells serveas environmental signals.,Growth Factor ST and Cancer,Growth factors regulate growth,proliferat
9、ion,and survival.These are all deregulated in cancer.,Hanahan and Weinberg,(2000)Hallmarks of Cancer,Cell(100)57,Growth factors with Oncogenic Potential,PDGF,originally shown to regulate proliferation,was also shown to have homology to v-sis,the simian sarcoma virus.Other viral oncogenes encoded pro
10、tein products that were growth factors that often overexpressed in cancer such as TGF-a.Autocrine signalling leads to deregulated growth.,PDGF familyNeurotrophinsA chainNGFB chain(c-sis)BDNFFGF FamilyNT3acidic FGFCytokines(Hematopoietic)basic FGFIL-2EGF Family IL-3EGFM-CSFTGF-aGM-CSF,GF Receptors wi
11、th Oncogenic Potential,EGFR,kinase activity stimulated by EGF-1 and TGF-a involved incell growth and differentiation,was linked via sequence homology to a known avian erythroblastosis virus onocgene,v-erbB.Since then,many oncogenes have been shown to encode for GFRs.,EGFR familyInsulin Receptor fami
12、lyerbB1(c-erbB)IGF-1(c-ros)erbB2(neu)Neurotrophins FGF FamilyNGFR(trk)FGFR-1(fig)BDNFR(trk-B)FGFR-2(K-sam)NT3 R(trk-C)PDGFR FamilyCSF-1R(c-fms)SLF R(c-kit),Induction of cancer by alternations in several types of proteins involved in cell growth control,Signal Transduction and Cancer,Lecture II:Intra
13、cellular SignallingOutline:What are some signalling pathways?What are their cell biological outputs?How do these result in the cancer phenotype?How can we exploit signalling pathways for therapy?,Generic Signal Transduction,RTK Signal Transduction,Signal TransductionDownstream effectors,Protein Sign
14、aling Modules(Domains),SH2 and PTB bind to tyrosine phosphorylated sitesSH3 and WW bind to proline-rich sequencesPDZ domains bind to hydrophobic residues at the C-termini of target proteinsPH domains bind to different phosphoinositidesFYVE domains specifically bind to Pdtlns(3)P(phosphatidylinositol
15、 3-phosphate),Mechanisms for Activation of Signaling Proteins by RTKs,Activation by membrane translocation,Activation by a conformational change,Activation by tyrosine phosphorylation,Mechanisms for Attenuation&Termination of RTK Activation,1)Ligand antagonists2)Receptor antagonists3)Phosphorylation
16、 and dephosphorylation4)Receptor endocytosis5)Receptor degradation by the ubiquitin-proteosome pathway,Activation of MAPK Pathways by Multiple Signals,Growth,differentiation,inflammation,apoptosis-tumorigenesis,Overview of MAPK Signaling Pathways,The MAPK Pathway Activated by RTK,P,RTK ST-PI3K pathw
17、ay,Proto-oncogenes that Encode for Signalling Proteins,Serine/Threonine Kinasesc-raf familyaktNon-receptor Tyrosine KinasessrcablReceptor associated binding proteinsc-ras family,Ras recruits Raf to the membrane,ST intermediates can be targets for anti-cancer drugs,Kinases:Raf,ST intermediates can be
18、 targets for anti-cancer drugs,Kinases:Bcr-Abl,Cell PatterningCell Growth,WntBMPHedgehogFGF,What are the essential elements of any Signaling cascade?,Signal ligandDiffusible or TetheredReceptor Transmembrane(except for lipid soluble ligands)Transducers-effectorsTargets Genes or Cellular components,W
19、nt Signaling Pathway,SignalWntsReceptor FrizzledsTransducers-effectorsb-cateninTargets Genes cytoskeleton,Wingless(Wg):Drosophila morphogen-diff.Concentrations of ligand elicit differentresponses in equivalent cells morphogenic movements and cell fate determinants“Be posterior”-cell fate“divide”-pro
20、liferation developmental abnormalities when gene deleted,The Signal:Wnt,Sharma describes a wingless mutation in 1973,Sharma,1973 Wingless-a new mutant in D.melanogaster.D.I.S.50:134 Sharma and Chopra,1976,Effect of the wingless(wg1)mutation on wing and haltere development in Drosophila melanogaster.
21、Dev.Biol.48:461-465,Later it was cloned positionally,Integration of MMTV causes mammary tumors in mice,Tumors are pregnancy dependentMMTV has a steroid enhancerMice develop breast tumors but only during lactationGene was designated-Int-1(integration of MMTV)Other insertion sites occurred at other GF
22、s e.g.FGFTumors exhibit dominant Gain of FunctionLesson:Ectopic activation of a gene hyperplasia=Oncogene,Wingless+int-1=Wnt,Fly wg and Mouse Int-1 are homologs,Genes are cloned.Sequence is similar 10 20 30 40 50 60 70 80 90 100H Wnt-1 MGLWALLPSWVSTTLLLALTALPAALAANS-SGR-WWGIVNIASSTNLLTDSKSLQLVLEPSLQ
23、LLSR-KQRRLIRQNPGILHSVSGGLQSAVFly Wg MDISYIFVICLMALCSGGSSLSQVEGKQKSGRGRGSMWWGIAKVGEPNNITP-IMYMDPAIHSTLRRKQRRLVRDNPGVLGALVKGANLAI 110 120 130 140 150 160 170 180 190 200H Wnt-1 RECKWQFRNRRWNCPT-APGPHLFGKIVNRGCRETAFIFAITSAGVTHSVARSCSEGSIESCTCDYRR-RGP-GGPDWHWGGCSDNIDFly Wg SECQHQFRNRRWNCSTRNFSRGKNLFGKIV
24、DRGCRETSFIYAITSAAVTHSIARACSEGTIESCTCDYSHQSRSPQANHQAGSVAGVRDWEWGGCSDNIG 210 220 230 240 250 260 270 280 290 300H Wnt-1 FGRLFGREFVDSGEKGRDLRFLMNLHNNEAGRTTVFSEMRQECKCHGMSGSCTVRTCWMRLPTLRAVGDVLRDRFDGASRVLYGN-Fly Wg FGFKFSREFVDTGERGRNLREKMNLHNNEAGRAHVQAEMRQECKCHGMSGSCTVKTCWMRLANFRVIGDNLKARFDGATRVQVTNSLRA
25、TNALAPVSPNA 310 320 330 340 350 360 370 380 390 400H Wnt-1 RGSN-RASR-AELLRLEPEDPAHKPPSPHDLVYFFly Wg AGSNSVGSNGLIIPQSGLVYGEEEERMLNDHMPDILLENSHPISKIHHPNMPSPNSLPQAGQRGGRNGRRQGRKHNRYHFQLNPHNPEHKPPGSKDLVYL 410 420 430 440 450 460 470 H Wnt-1 EKSPNFCTYSGRLGTAGTAGRACNSSSPALDGCELLCCGRGHRTRTQRVTERCNCTFHWCCHV
26、SCRNCTHTRVLHECLNFly Wg EPSPSFCEKNLRQGILGTHGRQCNETSLGVDGCGLMCCGRGYRRDEVVVVERCACTFHWCCEVKCKLCRTKKVIYTCLN,The Signal:Wnt,Secreted protein ligands of 80-100aa Lipid modifiedLatest breakthrough(2003):purification of active Wntrequires organic extraction!Short range acting Stick to extracellular matrix Gr
27、adients-morphogenic?multiple Wnts(19 in human/mouse and 7 in Drosophila),Wnts signal through serpentine receptors,2 classes of signaling receptorsCatalyticTyrosine Kinase Receptors RTKsSer/Thr Kinase Receptors BMPsSerpentine/G-protein-coupled-receptors(GPCRs)/7-transmembraneWntsadrenergic,dopamine,e
28、pinepherine etc,The Receptor:Frizzled,core receptor for Wnts seven-pass transmembrane proteins probably G-protein coupled receptors multiple Frizzleds(10 in human/mouse and 4 in Drosophila),a newly identified co-receptor for Wnts single pass transmembrane protein related to family of lipoprotein rec
29、eptors,LRP/arrow:,Wnt Signaling regulates gene expression and cell polarity,canonical,Lrp,non-canonical,Canonical Wnt signaling in 2005,http:/www.stanford.edu/rnusse/pathways/cell2/,b-catenin is the cytoplasmic-nuclear signaling mediator,b-catenin,b-catenin,armadillo in Drosophilagenetics determined
30、 that it functioned downstream of Wg b-catenin in mammalian system identified as componentof cell adhesion junctions subcellular localization of protein controversial for years purification of b-catenin and cloning of gene in 1991 by P.McCrae and B.Gumbiner showed that armadillo and b-catenin are or
31、thologues,The transducer/effector:,Armadillo repeat structure of b-catenin,CK1,E-cadherin,Wnt signaling pathway,C.Liu et al.2002.Cell 108:837.,Complicated phosphorylation controls b-catenin stability,How does b-catenin reach target genes?,LEF/TCF transcription factors HMG(High Mobility Group)protein
32、s mammalian LEF-1 and TCF-1 identified in T lymphocytesin 1991 two more members cloned by low stringency screening ofLibraries and degenerate PCR in 1993 b-catenin was used in a yeast two-hybrid assay andLEF-1 was cloned as an interacting protein in 1997-endpoint of the pathway determined-merged two
33、 independent groups of scientists-subcellular localization of b-catenin finally settled,General Structure of LEF/TCF Transcription Factors,b-catenin binding,Co-repressor binding,DNA binding/bending,alt.COOH,TCF-1,TCF-3,TCF-4,B,B,E,E,E,94%,96%,99%,55%,52%,64%,Target Genes of Wnt Signaling,cell cycle
34、regulators and transcription factors-c-MYC-cyclin D1 tissue specific genes tissue remodeling proteins-matrix metalloproteinases-ephrin receptors and ligands-adhesion proteins angiogenesis-VEGF,In the absence of Wnt signaling:,Groucho,GSK-3b,APC,Adenomatous Polyposis Coli Identified by Joanna Groden
35、and Ray White as a tumor suppressorgene suffering LOH in families with very high rates of colon cancer.Truncation mutations or loss of the entire gene occurs in mostSporadic colon cancers,http:/www.stanford.edu/rnusse/wntwindow.htm,Hereditary colorectal cancer(15%),Familial Adenomatous Polyposis(FAP
36、)-1%all colorectal CAGermline mutations in APC gene.Accelerated tumour initiation.Rate limiting step is somatic mutation in other APC alleleMedian age of cancer diagnosis 42 yrs.Despite shared genotypes,not all clinical disease is similar(disease modifying genes or environmental influences?)Often de
37、velop extracolonic manifestations.Mouse model APCminHereditary NonPolyposis Colorectal Cancer(HNPCC)-2-4%all colorectal CAMutations in DNA mismatch repair(MMR)genes-germline-genome-wide microsatellite instability(MI).Early clues came from bacterial studies.Adenomas form at the same rate as the gener
38、al population,but there is accelerated tumour progressionMedian age of cancer diagnosis also 42 yrs.,FAP HNPCC SporadicSporadicAdenomas Cancers,Incidence 1:7000 1:5001 in 21 in 20,APC mutation 90%80%80%(prevalance)(germline)(somatic)(somatic),MMR deficiency 90%3%13%(prevalance),MMR gene 70%?65%mutat
39、ions,APC shuttle mode-speculativel,Wnt signaling and colorectal cancer,Major function of APC is the regulation of celluar b-catenin levels.Activation of wnt pathway in colon cancer drives cell proliferationTcf-responsive genes:c-myc,cyclin D1,PPARd-fibronectin and matrilysin(an extracellular metallo
40、proteinase),CNS,Mutation cluster region-all result in protein truncation,Rac GEF,Gray bars-b-catenin binding sites.APC may play a role in cell-cell adhesion(Cadherins)Red bars-Axin/Conductin binding sites(lost in mutations)Red arrows-nuclear export signals.Mutant APC accumulates in the nucleusAsef b
41、inding activates Rac at membranes,inducing membrane rufflingtherefore possibly affecting cell motilityMT-microtubule binding site.APC is involved in linking microtubules to kinetochorestherefore mutations can contribute to genomic instability,b-catenin destruction complex,Axin and APC physically int
42、eract.APC mutations in colon CA lack Axinbinding sites.-b-catenin binds to APC.APC/Axin complex regulates GSK3b kinase activity.Binds to Axin.Therefore Axin may serve a scaffolding function.Axin and APC are also GSK3b substrates,and phosphorylation increasestheir ability to bind b-catenin.How wnt si
43、gnals inhibit GSK3b activity is unclear.Dishevelled is critical.Wnt signal results in dephosphorylation of AxinPP2A dephosphorylates Axin.Its catalytic subunit binds Axin while itsregulatory subunit binds APC.The regulatory subunit of PP2Ais mutated in a subset of colon CA.How is PP2A activity regul
44、ated?-Where is the intracellular localization of the destruction complex?,APC mutation,Wild type APC,APC mutations result in increased genomic instability,Mouse Model-APCmin,Multiple intestinal neoplasia(min).APC gene mutation.Truncated protein atcodon 850.Htz have increased propensity for tumors.Tu
45、mors acquire somatic mutation in wild type APC allele.Tumors located in upper GI tract(not colorectal).Genetic background of mouse influences tumor load(?modifiers).MOM-1-possibly secreted phospholipase A2.APC1638T lacks C-terminal domain that binds tubulin,EB1-like proteins.homozygous ES cells have
46、 high degree of chromosomal instabilitybut homozygous mice do NOT exhibit increased tumor susceptibilityCooperating Oncogenes.Cyclooxygenase 2:deletion of COX-2 gene suppresses intestinal polyposisin APCD716 mice.COX-2 levels are increased in premalignant polyps.But COX-2 is expressed in interstitia
47、l cells not intestinal epithelium.Smad4:deletion of Smad4 in APCD716 mice resulted in more aggressivetumors(compound htz mice).Highlights the role of TGF signal in tumor progression.DNA methyltransferase:compound htz have reduced polyp numbers(epigenetic events?).,Tumour Progression,TGF signaling mu
48、tationsreceptor II mutations detected in regions of high grade dysplasia but absent in adenomas.In tumours with microsatellite instability(MI)mutations correlate with progression of adenomas to cancermutations in TGF signaling components(e.g.,smad4)-non MI tumoraccelerate/worsen murine(APCmin)intest
49、inal cancer model,Cell-cell adhesive complex mutations-cadherins,b-catenins,others?,3.Metalloproteinase activation-matrilysin is a tcf-responsive gene,compaction of the early embryo,-morphogeneticmovement of cells-establishment ofcell fates,and polarity,loss of cell-cell and cell-matrix recognition,
50、tissue invasion motility,normal development,cancer progression,“epithelial mesenchymal”transition,Hedgehog Signaling Pathway,SignalHedgehogReceptor PatchedTransducers-effectorsCubitus InterruptusTargets Genes,Mutations in Hedgehog signaling in humans embryos yields cyclopia(a form of holoprosencepha
