《肿瘤细胞信号转导》PPT课件.ppt

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 Receptors Outline: What is Signal Transduction? What are Growth Factors? How do they contribute to normal ST? How is this ST deregulated in Cancer?,Lecture I: Growth Factors and Receptors What is Signal Transduction? Signal Transduction is the process by which a cell converts an extracellular signal into a response. Involved in: Cell-cell communication Cells response to environment Intracellular homeostatsis- internal communication,Generic Signalling Pathway,Signal Receptor (sensor) Transduction Cascade Targets Response,Altered Metabolism,Metabolic Enzyme,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,What 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, tastants,Signal = LIGAND Ligand- A molecule that binds to a specific site on 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,Intracellular Receptor,Nucleus,Adapted 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 linked receptors Signal diverse cellular responses including: Proliferation Differentiation Growth Survival Angiogenesis Can 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 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 serve as environmental signals.,Growth Factor ST and Cancer,Growth factors regulate growth, proliferation, 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 protein products that were growth factors that often overexpressed in cancer such as TGF-a. Autocrine signalling leads to deregulated growth.,PDGF family Neurotrophins A chain NGF B chain (c-sis) BDNF FGF Family NT3 acidic FGF Cytokines (Hematopoietic) basic FGF IL-2 EGF Family IL-3 EGF M-CSF TGF-a GM-CSF,GF Receptors with Oncogenic Potential,EGFR, kinase activity stimulated by EGF-1 and TGF-a involved in cell 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 family Insulin Receptor family erbB1 (c-erbB) IGF-1 (c-ros) erbB2 (neu) Neurotrophins FGF Family NGFR (trk) FGFR-1(fig) BDNFR (trk-B) FGFR-2(K-sam) NT3 R (trk-C) PDGFR Family CSF-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: Intracellular Signalling Outline: 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 Transduction Downstream effectors,Protein Signaling Modules (Domains),SH2 and PTB bind to tyrosine phosphorylated sites SH3 and WW bind to proline-rich sequences PDZ domains bind to hydrophobic residues at the C-termini of target proteins PH domains bind to different phosphoinositides FYVE domains specifically bind to Pdtlns(3)P (phosphatidylinositol 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 antagonists 2) Receptor antagonists 3) Phosphorylation and dephosphorylation 4) Receptor endocytosis 5) 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 pathway,Proto-oncogenes that Encode for Signalling Proteins,Serine/Threonine Kinases c-raf family akt Non-receptor Tyrosine Kinases src abl Receptor associated binding proteins c-ras family,Ras recruits Raf to the membrane,ST intermediates can be targets for anti-cancer drugs,Kinases: Raf,ST intermediates can be targets for anti-cancer drugs,Kinases: Bcr-Abl,Cell Patterning Cell Growth,Wnt BMP Hedgehog FGF,What 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 components,Wnt Signaling Pathway,Signal Wnts Receptor Frizzleds Transducers - effectors b-catenin Targets Genes cytoskeleton,Wingless (Wg): Drosophila morphogen - diff. Concentrations of ligand elicit different responses in equivalent cells morphogenic movements and cell fate determinants “Be posterior” - cell fate “divide” - proliferation 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. Dev. Biol. 48: 461-465,Later it was cloned positionally,Integration 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 Lesson: 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 90 100 H Wnt-1 MGLWALLPSWVSTTLLLALTALPAALAANS-SGR-WWGIVNIASSTNLLTDSKSLQLVLEPSLQLLSR-KQRRLIRQNPGILHSVSGGLQSAV Fly Wg MDISYIFVICLMALCSGGSSLSQVEGKQKSGRGRGSMWWGIAKVGEPNNITP-IMYMDPAIHSTLRRKQRRLVRDNPGVLGALVKGANLAI 110 120 130 140 150 160 170 180 190 200 H Wnt-1 RECKWQFRNRRWNCPT-APGPHLFGKIVNRGCRETAFIFAITSAGVTHSVARSCSEGSIESCTCDYRR-RGP-GGPDWHWGGCSDNID Fly Wg SECQHQFRNRRWNCSTRNFSRGKNLFGKIVDRGCRETSFIYAITSAAVTHSIARACSEGTIESCTCDYSHQSRSPQANHQAGSVAGVRDWEWGGCSDNIG 210 220 230 240 250 260 270 280 290 300 H Wnt-1 FGRLFGREFVDSGEKGRDLRFLMNLHNNEAGRTTVFSEMRQECKCHGMSGSCTVRTCWMRLPTLRAVGDVLRDRFDGASRVLYGN- Fly Wg FGFKFSREFVDTGERGRNLREKMNLHNNEAGRAHVQAEMRQECKCHGMSGSCTVKTCWMRLANFRVIGDNLKARFDGATRVQVTNSLRATNALAPVSPNA 310 320 330 340 350 360 370 380 390 400 H Wnt-1 RGSN-RASR-AELLRLEPEDPAHKPPSPHDLVYF Fly Wg AGSNSVGSNGLIIPQSGLVYGEEEERMLNDHMPDILLENSHPISKIHHPNMPSPNSLPQAGQRGGRNGRRQGRKHNRYHFQLNPHNPEHKPPGSKDLVYL 410 420 430 440 450 460 470 H Wnt-1 EKSPNFCTYSGRLGTAGTAGRACNSSSPALDGCELLCCGRGHRTRTQRVTERCNCTFHWCCHVSCRNCTHTRVLHECLN Fly Wg EPSPSFCEKNLRQGILGTHGRQCNETSLGVDGCGLMCCGRGYRRDEVVVVERCACTFHWCCEVKCKLCRTKKVIYTCLN,The Signal: Wnt, Secreted protein ligands of 80-100aa Lipid modified Latest breakthrough (2003): purification of active Wnt requires organic extraction! Short range acting Stick to extracellular matrix Gradients - morphogenic? multiple Wnts (19 in human/mouse and 7 in Drosophila),Wnts signal through serpentine receptors,2 classes of signaling receptors Catalytic Tyrosine Kinase Receptors RTKs Ser/Thr Kinase Receptors BMPs Serpentine/G-protein-coupled-receptors(GPCRs) /7-transmembrane Wnts adrenergic, dopamine, epinepherine 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 receptors,LRP/arrow:,Wnt Signaling regulates gene expression and cell polarity,canonical,Lrp,non-canonical,Canonical Wnt signaling in 2005,/rnusse/pathways/cell2/,/cgi/cm/CMP_5533,b-catenin is the cytoplasmic-nuclear signaling mediator,b-catenin,b-catenin, armadillo in Drosophila genetics determined that it functioned downstream of Wg b-catenin in mammalian system identified as component of 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 orthologues,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) proteins mammalian LEF-1 and TCF-1 identified in T lymphocytes in 1991 two more members cloned by low stringency screening of Libraries and degenerate PCR in 1993 b-catenin was used in a yeast two-hybrid assay and LEF-1 was cloned as an interacting protein in 1997 - endpoint of the pathway determined - merged two 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 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 and Ray White as a tumor suppressor gene suffering LOH in families with very high rates of colon cancer. Truncation mutations or loss of the entire gene occurs in most Sporadic colon cancers ,/rnusse/wntwindow.htm,Hereditary colorectal cancer ( 15%),Familial Adenomatous Polyposis (FAP) - 1% all colorectal CA Germline mutations in APC gene. Accelerated tumour initiation. Rate limiting step is somatic mutation in other APC allele Median age of cancer diagnosis 42 yrs. Despite shared genotypes, not all clinical disease is similar (disease modifying genes or environmental influences?) Often develop extracolonic manifestations. Mouse model APCmin Hereditary NonPolyposis Colorectal Cancer (HNPCC) - 2-4% all colorectal CA Mutations 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 general population, but there is accelerated tumour progression Median age of cancer diagnosis also 42 yrs.,FAP HNPCC Sporadic Sporadic Adenomas Cancers,Incidence 1:7000 1:500 1 in 2 1 in 20,APC mutation 90% 80% 80% (prevalance) (germline) (somatic) (somatic),MMR deficiency 90% 3% 13% (prevalance),MMR gene 70% ? 65% mutations,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 proliferation Tcf-responsive genes: c-myc, cyclin D1, PPARd - fibronectin and matrilysin (an extracellular metalloproteinase),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 nucleus Asef binding activates Rac at membranes, inducing membrane ruffling therefore possibly affecting cell motility MT - microtubule binding site. APC is involved in linking microtubules to kinetochores therefore mutations can contribute to genomic instability,b-catenin 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 GSK3b 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 dephosphorylation 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 mutation,Wild type APC,APC mutations result in increased genomic instability,Mouse Model - APCmin,Multiple intestinal neoplasia (min). APC gene mutation. Truncated protein at codon 850. Htz have increased propensity for tumors. Tumors 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 high degree of chromosomal instability but homozygous mice do NOT exhibit increased tumor susceptibility Cooperating Oncogenes. Cyclooxygenase 2: deletion of COX-2 gene suppresses intestinal polyposis in 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 in more aggressive tumors (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 mutations receptor 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 cancer mutations in TGF signaling components (e.g., smad4) - non MI tumor accelerate/worsen murine (APCmin) intestinal 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,- morphogenetic movement of cells - establishment of cell fates, and polarity,loss of cell-cell and cell-matrix recognition,tissue invasion motility,normal development,cancer progression,“epithelial mesenchymal” transition,Hedgehog Signaling Pathway,Signal Hedgehog Receptor Patched Transducers - effectors Cubitus Interruptus Targets 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 stem 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: Hedgehog, 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 tissues. 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 fragment becomes tethered to the membrane via a hydrophobic cholesterol moiety (it doesnt contain any hydrophobic residues). A series of elegant experiments show that this arrangement ensures that hedgehog only acts on the adjacent cell and doesnt diffuse to act on cells further away (i.e. it is spatially restricted).,The Signal: Hedgehog,Hedgeh