腫瘤細胞信號轉導ppt課件

1、如何閱讀文獻 在腫瘤學方面有哪些好的雜志 如何快速閱讀文獻以獲取知識 論文的主要結構腫瘤的分子信號轉導 + genomic instabilityfrom Hanahan and Weinberg 2000Signal Transduction and CancerLecture 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 Signa

3、lling PathwaySignalReceptor (sensor)Transduction CascadeTargetsResponse Altered MetabolismMetabolicEnzymeGene RegulatorCytoskeletal ProteinAltered Gene ExpressionAltered Cell Shape or MotilityAdapted from Molecular Biology of the Cell,(2019), 4th edition, Alberts et al. Components of SignallingWhat

4、can be the Signal?External message to the cell Peptides / Proteins- Growth Factors Amino acid derivatives - epinephrine, histamine Other small biomolecules - ATP Steroids, prostaglandins Gases - Nitric Oxide (NO) Photons Damaged DNA Odorants, tastantsSignal = LIGANDLigand- A molecule that binds to a

5、 specific site on another molecule, usually a protein, ie receptorComponents of SignallingWhat are Receptors?Sensors, what the signal/ligand binds to initiate ST Cell surface Intracellular Hydrophillic LigandCell-Surface ReceptorPlasma membraneHydrophobic LigandCarrier ProteinIntracellularReceptorNu

6、cleusAdapted from Molecular Biology of the Cell,(2019), 4th edition, Alberts et al. Cell Surface Receptor Types:Ligand-gated ion channelCell Surface Receptor Types:2) G-Protein Coupled ReceptorCell Surface Receptor Types:3) Enzyme-linked Receptor eg Growth Factor ReceptorsGrowth FactorsLigands which

7、 bind enzyme linked receptorsSignal diverse cellular responses including:ProliferationDifferentiationGrowthSurvival AngiogenesisCan signal to multiple cell types or be specificFactorPrincipal SourcePrimary ActivityCommentsPDGFplatelets, endothelial cells, placentapromotes proliferation of connective

8、 tissue, glial and smooth muscle cellstwo different protein chains form 3 distinct dimer forms; AA, AB and BBEGFsubmaxillary gland, Brunners glandpromotes proliferation of mesenchymal, glial and epithelial cells TGF-common in transformed cellsmay be important for normal wound healingrelated to EGFFG

9、Fwide range of cells; protein is associated with the ECMpromotes proliferation of many cells; inhibits some stem cells; induces mesoderm to form in early embryosat least 19 family members, 4 distinct receptorsNGF promotes neurite outgrowth and neural cell survivalseveral related proteins first ident

10、ified as proto-oncogenes; trkA (trackA), trkB, trkCErythropoietinkidneypromotes proliferation and differentiation of erythrocytes TGF-activated TH1 cells (T-helper) and natural killer (NK) cellsanti-inflammatory (suppresses cytokine production and class II MHC expression), promotes wound healing, in

11、hibits macrophage and lymphocyte proliferationat least 100 different family membersIGF-Iprimarily liverpromotes proliferation of many cell typesrelated to IGF-II and proinsulin, also called Somatomedin CIGF-IIvariety of cellspromotes proliferation of many cell types primarily of fetal originrelated

12、to IGF-I and proinsulinGrowth Factor ReceptorsMost growth factors bind Receptor Tyrosine KinasesCharacteristics of the Common Classes ofRTKsClassExamplesStructural Features of ClassIEGF receptor,NEU/HER2, HER3cysteine-rich sequencesIIinsulin receptor,IGF-1 receptorcysteine-rich sequences; characteri

13、zed bydisulfide-linked heterotetramersIIIPDGF receptors,c-Kitcontain 5 immunoglobulin-like domains;contain the kinase insertIVFGF receptorscontain 3 immunoglobulin-like domains aswell as the kinase insert; acidic domainVvascularendothelial cellgrowth factor(VEGF) receptorcontain 7 immunoglobulin-lik

14、e domains aswell as the kinase insert domainVIhepatocyte growthfactor (HGF) andscatter factor (SC)receptorsheterodimeric like the class II receptorsexcept that one of the two protein subunitsis completely extracellular. The HGFreceptor is a proto-oncogene that wasoriginally identified as the Met onc

15、ogeneVIIneurotrophinreceptor family(trkA, trkB, trkC)and NGF receptorcontain no or few cysteine-rich domains;NGFR has leucine rich domainGrowth Factor Receptor Activation IRTKRS/TKGrowth Factor Receptor Activation IIGrowth signal autonomy,Insensitivity to anti-growth signals,Resistance to apoptosis:

16、Uncouple cells growth program from signals in the environment.Growth factors in normal cells serveas environmental signals. Growth Factor ST and CancerGrowth factors regulate growth, proliferation, and survival.These are all deregulated in cancer.Hanahan and Weinberg, (2000) Hallmarks of Cancer, Cel

17、l (100) 57Growth factors with Oncogenic PotentialPDGF, originally shown to regulate proliferation, was also shown to have homology to v-sis, the simian sarcoma virus. Other viral oncogenes encoded protein products that were growth factors that often overexpressed in cancer such as TGF-a. Autocrine s

18、ignalling leads to deregulated growth. PDGF familyNeurotrophinsA chainNGFB chain (c-sis)BDNFFGF FamilyNT3acidic FGFCytokines (Hematopoietic)basic FGFIL-2EGF Family IL-3EGFM-CSFTGF-aGM-CSFGF Receptors with Oncogenic PotentialEGFR, kinase activity stimulated by EGF-1 and TGF-a involved incell growth a

19、nd 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 familyerbB1 (c-erbB)IGF-1 (c-ros)erbB2 (neu) Neurotrophins FGF FamilyNGFR (trk) FGFR-1(fig)BDNFR (t

20、rk-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 CancerLecture II: Intracellular SignallingOutline:What are some signalling pathways?What are their cell biolo

21、gical outputs?How do these result in the cancer phenotype?How can we exploit signalling pathways for therapy?Generic Signal TransductionRTK Signal TransductionSignal TransductionDownstream effectorsProtein Signaling Modules (Domains)SH2 and PTB bind to tyrosine phosphorylated sitesSH3 and WW bind to

22、 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 3-phosphate)Mechanisms for Activation of Signaling Proteins by RTKsActivation by membran

23、e translocationActivation by a conformational changeActivation by tyrosine phosphorylationMechanisms for Attenuation & Termination of RTK Activation1) Ligand antagonists2) Receptor antagonists3) Phosphorylation and dephosphorylation4) Receptor endocytosis5) Receptor degradation by the ubiquitin-

24、proteosome pathwayActivation of MAPK Pathways by Multiple SignalsGrowth, differentiation, inflammation, apoptosis - tumorigenesisOverview of MAPK Signaling PathwaysThe MAPK Pathway Activated by RTKPRTK ST- PI3K pathwayProto-oncogenes that Encode for Signalling ProteinsSerine/Threonine Kinasesc-raf f

25、amilyaktNon-receptor Tyrosine KinasessrcablReceptor associated binding proteinsc-ras familyRas recruits Raf to the membraneST intermediates can be targets for anti-cancer drugsKinases:RafST intermediates can be targets for anti-cancer drugsKinases:Bcr-AblCell PatterningCell GrowthWntBMPHedgehogFGFWh

26、at are the essential elements of any Signaling cascade? Signal ligand Diffusible or Tethered Receptor Transmembrane (except for lipid soluble ligands) Transducers - effectors Targets Genes or Cellular componentsWnt Signaling PathwaySignalWntsReceptor FrizzledsTransducers - effectorsb-cateninTargets

27、Genes cytoskeletonWingless (Wg): Drosophila morphogen - diff. Concentrations of ligand elicit differentresponses in equivalent cells morphogenic movements and cell fate determinants “Be posterior” - cell fate “divide” - proliferation developmental abnormalities when gene deletedThe Signal: WntSharma

28、 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. Dev. Biol. 48: 461-465Later it was cloned positionallyIntegratio

29、n of MMTV causes mammary tumors in mice Tumors are pregnancy dependent MMTV has a steroid enhancer Mice develop breast tumors but only during lactation Gene was designated - Int-1 (integration of MMTV) Other insertion sites occurred at other GFs e.g. FGF Tumors exhibit dominant Gain of Function Less

30、on: Ectopic activation of a gene hyperplasia = OncogeneWingless + int-1 = WntFly wg and Mouse Int-1 are homologs Genes are cloned. Sequence is similar 10 20 30 40 50 60 70 80 90 100 H Wnt-1 MGLWALLPSWVSTTLLLALTALPAALAANS-SGR-WWGIVNIASSTNLLTDSKSLQLVLEPSLQLLSR-KQRRLIRQNPGILHSVSGGLQSAV Fly Wg MDISYIFVI

31、CLMALCSGGSSLSQVEGKQKSGRGRGSMWWGIAKVGEPNNITP-IMYMDPAIHSTLRRKQRRLVRDNPGVLGALVKGANLAI 110 120 130 140 150 160 170 180 190 200 H Wnt-1 RECKWQFRNRRWNCPT-APGPHLFGKIVNRGCRETAFIFAITSAGVTHSVARSCSEGSIESCTCDYRR-RGP-GGPDWHWGGCSDNID Fly Wg SECQHQFRNRRWNCSTRNFSRGKNLFGKIVDRGCRETSFIYAITSAAVTHSIARACSEGTIESCTCDYSHQSR

32、SPQANHQAGSVAGVRDWEWGGCSDNIG 210 220 230 240 250 260 270 280 290 300 H Wnt-1 FGRLFGREFVDSGEKGRDLRFLMNLHNNEAGRTTVFSEMRQECKCHGMSGSCTVRTCWMRLPTLRAVGDVLRDRFDGASRVLYGN- Fly Wg FGFKFSREFVDTGERGRNLREKMNLHNNEAGRAHVQAEMRQECKCHGMSGSCTVKTCWMRLANFRVIGDNLKARFDGATRVQVTNSLRATNALAPVSPNA 310 320 330 340 350 360 370 3

33、80 390 400 H Wnt-1 RGSN-RASR-AELLRLEPEDPAHKPPSPHDLVYF Fly Wg AGSNSVGSNGLIIPQSGLVYGEEEERMLNDHMPDILLENSHPISKIHHPNMPSPNSLPQAGQRGGRNGRRQGRKHNRYHFQLNPHNPEHKPPGSKDLVYL 410 420 430 440 450 460 470 H Wnt-1 EKSPNFCTYSGRLGTAGTAGRACNSSSPALDGCELLCCGRGHRTRTQRVTERCNCTFHWCCHVSCRNCTHTRVLHECLN Fly Wg EPSPSFCEKNLRQGI

34、LGTHGRQCNETSLGVDGCGLMCCGRGYRRDEVVVVERCACTFHWCCEVKCKLCRTKKVIYTCLNThe Signal: Wnt Secreted protein ligands of 80-100aa Lipid modifiedLatest breakthrough (2019): purification of active Wntrequires organic extraction! Short range acting Stick to extracellular matrix Gradients - morphogenic? multiple Wnt

35、s (19 in human/mouse and 7 in Drosophila)Wnts signal through serpentine receptors2 classes of signaling receptorsCatalyticTyrosine Kinase Receptors RTKsSer/Thr Kinase Receptors BMPsSerpentine/G-protein-coupled-receptors(GPCRs) /7-transmembraneWntsadrenergic, dopamine, epinepherine etcThe Receptor: F

36、rizzled 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 receptorsLRP/arrow:Wnt Signaling

37、 regulates gene expression and cell polaritycanonicalWntFzWntFzLrpLrpnon-canonicalCanonical Wnt signaling in 2019/rnusse/pathways/cell2//cgi/cm/CMP_5533-ctenin is the cytoplsmic-nucler signling meditor-ctenin is the cytoplsmic-nucler signling meditor-ctenin-ctenin-cten

38、in-ctenin armadillo in Drosophilagenetics determined 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

39、 B. Gumbiner showed that armadillo and b-catenin are orthologuesThe transducer/effector:Armadillo repeat structure of b-cateninLEF/TCFWntCK1GSK-3bAPC-ctenin-cteninaxinFrizzledLRPE-cadherinWnt signaling pathwayC. Liu et al. 2019. Cell 108:837.Complicated phosphorylation controls b-catenin stabilityHo

40、w does b-catenin reach target genes? LEF/TCF transcription factors HMG (High Mobility Group) proteins 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

41、 andLEF-1 was cloned as an interacting protein in 2019- endpoint of the pathway determined- merged two independent groups of scientists- subcellular localization of b-catenin finally settledGeneral Structure of LEF/TCF Transcription Factors-ctenin -ctenin indingindingCo-repressor bindingDNA binding/

42、bendingalt.COOHTCF-1TCF-3TCF-4NLSHMGNLEF-1BBBEEE94%96%99%55%52%64%Target Genes of Wnt Signaling cell cycle regulators and transcription factors-c-MYC-cyclin D1 tissue specific genes tissue remodeling proteins- matrix metalloproteinases- ephrin receptors and ligands- adhesion proteins angiogenesis- V

43、EGFIn the absence of Wnt signaling:NLSHMGNLEF-1BNLSHMGNdnLEF-1BGrouchoLEF/TCFWntGSK-3baxinFrizzledLRPAPCLEF/TCFLEF/TCFLEF/TCFLEF/TCFLEF/TCFLEF/TCFLEF/TCFLEF/TCFActivation of LEF-1 target genes can transform cellsAoki, M. et al. 2019. Proc. Natl. Acad. Sci. USA. 96:139-144anchorage-independent growth

44、 contact inhibition colony formationAdenomatous Polyposis Coli Identified by Joanna Groden 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 /rnusse/

45、wntwindow.htmHereditary colorectal cancer ( 15%)Familial Adenomatous Polyposis (FAP) - 90% 80% 80% (prevalance) (germline)(somatic) (somatic)MMR deficiency 90% 70% ? 65% mutationsAPC shuttle mode - speculativel Wnt signaling and colorectal cancerMajor function of APC is the regulation of celluar b-c

46、atenin levels.Activation of wnt pathway in colon cancer drives cell proliferationTcf-responsive genes: c-myc, cyclin D1, PPARd- fibronectin and matrilysin (an extracellular metalloproteinase)CNSMutation cluster region - all result in protein truncationRac GEFGray bars - b-catenin binding sites. APC

47、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 binding activates Rac at membranes, inducing membrane rufflingtherefore possibly affecting cell motilityMT - micr

48、otubule binding site. APC is involved in linking microtubules to kinetochorestherefore mutations can contribute to genomic instability-ctenin destruction complex-Axin and APC physically interact. APC mutations in colon CA lack Axin-binding sites.- b-catenin binds to APC.-APC/Axin complex regulates G

49、SK3b kinase activity. Binds to Axin.-Therefore Axin may serve a scaffolding function.-Axin and APC are also GSK3b substrates, and phosphorylation increases-their ability to bind b-catenin.-How wnt signals inhibit GSK3b activity is unclear. Dishevelled is critical.-Wnt signal results in dephosphoryla

50、tion of Axin-PP2A dephosphorylates Axin. Its catalytic subunit binds Axin while its-regulatory subunit binds APC. The regulatory subunit of PP2A-is mutated in a subset of colon CA. How is PP2A activity regulated?- Where is the intracellular localization of the destruction complex?APC mutationWild ty

51、pe APCAPC mutations result in increased genomic instabilityMouse Model - APCminMultiple intestinal neoplasia (min). APC gene mutation. Truncated protein atcodon 850.Htz have increased propensity for tumors. Tumors acquire somatic mutation in wild type APC allele.Tumors located in upper GI tract (not

52、 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 high degree of chromosomal instabilitybut homozygous mice do NOT exhibit increased tu

53、mor 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 interstitial cells not intestinal epithelium.Smad4: deletion of Smad4 in APCD716 mice resulted

54、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 ProgressionTGF signaling mutationsreceptor II mutations detected in regions of high grade dysplasia but absen

55、t 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) intestinal cancer modelCell-cell adhesive complex mutations - cadherins, b-caten

56、ins, others?3. Metalloproteinase activation - matrilysin is a tcf-responsive gene compaction of the early embryo- morphogeneticmovement of cells- establishment ofcell fates, and polarityloss of cell-cell and cell-matrix recognitiontissue invasion motility“epithelial mesenchymal” transitionHedgehog S

57、ignaling PathwaySignalHedgehogReceptor PatchedTransducers - effectorsCubitus InterruptusTargets Genes Mutations in Hedgehog signaling in humans embryos yields cyclopia(a form of holoprosencephaly) images are only for the stout-hearted. In adults, mutations in Hedgehog signaling gives phenotypes in s

58、tem cell and progenitor populations . Increased signaling gives tumors, less signaling gives short-lived stem cells Most recent advance is that many tumors show elevated Hh signaling. Cyclopamine (first obtained from the corn lily) has promise for therapeutic intervention of cancer.The Signal: Hedge

59、hog Secreted protein ligand - heavily processed Lipid modified with cholesterol Short range acting Stick to extracellular matrix Gradients - morphogenic? three ligands in mammals: Indian, Desert, Sonic The hedgehog gene encodes a novel membrane-linked ligand important for local patterning of many ti

60、ssues. The primary translation product contains a signal peptide that is cleaved to produce a 45 kDa polypeptide precursor. Cleavage of this secreted precursor produces a 20 kDa N-terminal fragment associated with the plasma membrane and with inductive activity plus a 25 kDa fragment. The N-terminal fra