肿瘤细胞信号转导

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 instabilityfrom hanahan and weinberg 2000signal transduction and cancerlecture i: growth factors and receptorsoutline:a) what is signal transduction?b) what are growth factors?c) how do they contribute to normal st?d) how is this st dereg

2、ulated in cancer?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 g

3、eneric signalling pathwaysignalreceptor (sensor)transduction cascadetargetsresponse altered metabolismmetabolicenzymegene regulatorcytoskeletal proteinaltered gene expressionaltered cell shape or motilityadapted from molecular biology of the cell,(2002), 4th edition, alberts et al. components of sig

4、nallingwhat 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 tha

5、t binds to a 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 proteinintracellul

6、arreceptornucleusadapted from molecular biology of the cell,(2002), 4th edition, alberts et al. cell surface receptor types:1) ligand-gated ion channelcell surface receptor types:2) g-protein coupled receptorcell surface receptor types:3) enzyme-linked receptor eg growth factor receptorsgrowth facto

7、rsligands which bind enzyme linked receptorssignal diverse cellular responses including:proliferationdifferentiationgrowthsurvival angiogenesiscan signal to multiple cell types or be specificfactorprincipal sourceprimary activitycommentspdgfplatelets, endothelial cells, placentapromotes proliferatio

8、n of connective 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 healingr

9、elated to egffgfwide 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 prote

10、ins first identified 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 wo

11、und healing, inhibits 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

12、 originrelated 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 sequen

13、ces; characterized 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 imm

14、unoglobulin-like 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

15、 as the met oncogeneviineurotrophinreceptor 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,resistanc

16、e to apoptosis: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

17、 of cancer, cell (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

18、-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-csfgf receptors with oncogenic potentialegfr, kinase activity stimulated by egf-1 and tgf-a involved

19、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 familyerbb1 (c-erbb)igf-1 (c-ros)erbb2 (neu) neurotrophins fgf familyngfr (trk) fgfr

20、-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 cancerlecture ii: intracellular signallingoutline:a) what are some signalling pathways?b) what

21、 are their cell biological outputs?c) how do these result in the cancer phenotype?d) how can we exploit signalling pathways for therapy?generic signal transductionrtk signal transductionsignal transductiondownstream effectorsprotein signaling modules (domains)sh2 and ptb bind to tyrosine phosphoryla

22、ted 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 3-phosphate)mechanisms for activation of signaling proteins b

23、y rtksactivation by membrane translocationactivation by a conformational changeactivation by tyrosine phosphorylationmechanisms for attenuation & termination of rtk activation1) ligand antagonists2) receptor antagonists3) phosphorylation and dephosphorylation4) receptor endocytosis5) receptor de

24、gradation by the ubiquitin-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 proteinsseri

25、ne/threonine kinasesc-raf familyaktnon-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 patterningcel

26、l growthwntbmphedgehogfgfwhat 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 -

27、 effectors -catenintargets 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 d

28、eletedthe signal: wntsharma 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 cl

29、oned positionallyintegration 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 dom

30、inant gain of function lesson: 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 100h wnt-1 mglwallpswvsttlllaltalpaalaans-sgr-wwgivniasstnlltdskslqlvlepslqllsr-kqrrlirqnpgilhs

31、vsgglqsavfly wg mdisyifviclmalcsggsslsqvegkqksgrgrgsmwwgiakvgepnnitp-imymdpaihstlrrkqrrlvrdnpgvlgalvkganlai 110 120 130 140 150 160 170 180 190 200h wnt-1 reckwqfrnrrwncpt-apgphlfgkivnrgcretafifaitsagvthsvarscsegsiesctcdyrr-rgp-ggpdwhwggcsdnidfly wg secqhqfrnrrwncstrnfsrgknlfgkivdrgcretsfiyaitsaavth

32、siaracsegtiesctcdyshqsrspqanhqagsvagvrdwewggcsdnig 210 220 230 240 250 260 270 280 290 300h wnt-1 fgrlfgrefvdsgekgrdlrflmnlhnneagrttvfsemrqeckchgmsgsctvrtcwmrlptlravgdvlrdrfdgasrvlygn-fly wg fgfkfsrefvdtgergrnlrekmnlhnneagrahvqaemrqeckchgmsgsctvktcwmrlanfrvigdnlkarfdgatrvqvtnslratnalapvspna 310 320

33、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 ekspnfctysgrlgtagtagracnssspaldgcellccgrghrtrtqrvtercnctfhwcchvscrncthtrvlheclnfly

34、wg epspsfceknlrqgilgthgrqcnetslgvdgcglmccgrgyrrdevvvvercactfhwccevkcklcrtkkviytclnthe 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 gradients - morphog

35、enic? multiple wnts (19 in human/mouse and 7 in drosophila)wnts signal through serpentine receptors2 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, epineph

36、erine etcthe 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 receptor

37、slrp/arrow:wnt signaling regulates gene expression and cell polaritycanonicalwntfzwntfzlrplrpnon-canonicalcanonical wnt signaling in 2005/rnusse/pathways/cell2//cgi/cm/cmp_5533 -catenin is the cytoplasmic-nuclear signaling mediator -catenin -catenin arm

38、adillo in drosophilagenetics determined that it functioned downstream of wg -catenin in mammalian system identified as componentof cell adhesion junctions subcellular localization of protein controversial for years purification of -catenin and cloning of gene in 1991 by p. mccrae and b. gumbiner sho

39、wed that armadillo and -catenin are orthologuesthe transducer/effector:armadillo repeat structure of -cateninlef/tcfwntck1gsk-3 apc -cateninaxinfrizzledlrpe-cadherinwnt signaling pathwayc. liu et al. 2002. cell 108:837.complicated phosphorylation controls -catenin stabilityhow does -catenin reach ta

40、rget 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 -catenin was used in a yeast two-hybrid assay andlef-1 was cloned as an

41、 interacting protein in 1997- endpoint of the pathway determined- merged two independent groups of scientists- subcellular localization of -catenin finally settledgeneral structure of lef/tcf transcription factors -catenin bindingco-repressor bindingdna binding/bendingalt.coohtcf-1tcf-3tcf-4nlshmgnl

42、ef-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- vegfin the absence of wnt signaling:nls

43、hmgnlef-1bnlshmgndnlef-1bgroucholef/tcfwntgsk-3 axinfrizzledlrpapclef/tcflef/tcflef/tcflef/tcflef/tcflef/tcflef/tcflef/tcfactivation of lef-1 target genes can transform cellsaoki, m. et al. 1999. proc. natl. acad. sci. usa. 96:139-144anchorage-independent growth contact inhibition colony formationad

44、enomatous 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/wntwindow.htmhereditary colo

45、rectal 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 -catenin levels.activation of w

46、nt 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 - -catenin binding sites. apc may play a role in cell-cell

47、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 - microtubule binding site. apc is

48、involved in linking microtubules to kinetochorestherefore mutations can contribute to genomic instability-catenin destruction complex-axin and apc physically interact. apc mutations in colon ca lack axinbinding sites.- -catenin binds to apc.-apc/axin complex regulates gsk3 kinase activity. binds to

49、axin.therefore axin may serve a scaffolding function.-axin and apc are also gsk3 substrates, and phosphorylation increasestheir ability to bind -catenin.-how wnt signals inhibit gsk3 activity is unclear. dishevelled is critical.-wnt signal results in dephosphorylation of axin-pp2a dephosphorylates a

50、xin. 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 regulated?- where is the intracellular localization of the destruction complex?apc mutationwild type apcapc mutations result in increase

51、d 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 colorectal).genetic background of mou

52、se 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 tumor susceptibilitycooperating oncogene

53、s.1.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.2.smad4: deletion of smad4 in apcd716 mice resulted in more aggressivetumors (compound

54、 htz mice). highlights the role of tgf signal in tumor progression.3.dna methyltransferase: compound htz have reduced polyp numbers(epigenetic events?).tumour progression1.tgf signaling mutations-receptor ii mutations detected in regions of high grade dysplasia but absent in adenomas. in tumours wit

55、h microsatellite instability (mi)-mutations correlate with progression of adenomas to cancer-mutations in tgf signaling components (e.g., smad4) - non mi tumoraccelerate/worsen murine (apcmin) intestinal cancer model2.cell-cell adhesive complex mutations - cadherins, -catenins, others?3. metalloprot

56、einase 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 signaling pathwaysignalhedg

57、ehogreceptor 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 stem cell and progenitor po

58、pulations . 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 ligan

59、d - 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 transla

60、tion 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