resources肿瘤问题

,*,*,*,ZYY,制作,顾 健 人,上海市肿瘤研究所,癌基因及相关基因国家重点实验室,1,1.,现有“生物信息学”的限制：,“有眼不识泰山”,第一外显子 识别能力,外显子内含子 识别能力,2.,一个基因：不同剪切体,Splicing form (,Isoform,),3.,一个基因, 蛋白质 切割：,蛋白质 ,1,肽段,人,基因组计划已经给了我们多少？,人,基因有多少个？,10,万,3,万？,2,4.,同一个蛋白质,细胞内的不同 “定位” 不同功能！,5.,98,DNA,序列：,功能不明,!,（,95,）,细胞核,细胞质,线粒体,细胞膜,蛋白质移位,Translocation,3,E1,b,E1,a,Exon,2,Exon,3,p16,Ink 4a,P14 ARF (human),P19 ARF (mouse),Cross Talk,betweren,Oncogenes,& tumor suppressors,Oncogenic,Signals,Ras,c-myc, E2F,p16,Ink 4a,Rb,on,Cell cycle,arrest,P14,ARF,(Block MDM-2 / p53 binding),p53,on,DNA,Damage,一个基因可编编码两种完全不同的蛋白质,4,功能基因组学,Functional Genomics,阐明基因（蛋白质）之间的相互作用,与生命活动的关系,与疾病的关系,与环境、药物的关系,基因组测序只是基因组研究的起点,“功能基因组学”的含义被“局限地”误解,“基因表达谱”的误解或局限性,cDNA,芯片：,mRNA,表达,蛋白质表达,蛋白质芯片：只知道蛋白质的表达与存在,并不知道蛋白质的功能状态,问题：,在基因组的水平,5,1.,糖基化,(1),膜表面蛋白：影响细胞识别，细胞细胞相互关系,(2),细胞表面抗原：,(3),细胞内蛋白：,特定糖基化与蛋白质叠成为高级结构有关,成熟蛋白质不成熟蛋白质的“质控”表志,2.,乙酰化：,组蛋白：与,DNA,结合能力、复制、转录,其它蛋白质 （如,p53,）,3.,磷酸化：,4.,蛋白质切割成片段：,一个蛋白质多种功能。,6,细胞分子细胞生物学重要问题,过度增殖： 恶性肿瘤,高血压（内皮细胞、平滑肌细胞）,细胞凋亡： 神经退行行病变,早老性痴呆,帕金森氏病,“,To be or not to be, thats a question”,Hamlet,Shakespear,细胞,增殖,细胞,凋亡,生 死,7,细胞,凋亡基因,Apoptotic gene,原癌,基因,Proto-,oncogene,抑癌,基因,Cancer,Suppressor,DNA,复制基因,细胞周期,基因,DNA,转录,（抗,凋亡）,Antiapoptotic,（促进凋亡）,M,Check Points of Cell Cycle,G,1, S check point,G,2, M check point,M: Mitotic Spindle checkpoint,8,分子 细胞 组织 机体,误 区：,相互分离。每种疾病均集中于反映出疾病的细胞,肿 瘤：,集中在癌细胞,误 区：,忽视癌细胞以外的细胞,心血管疾病：,心脏病 心肌细胞 心肌间质血管内皮细胞,高血压 内皮细胞 内皮细胞间质细胞炎症细胞 平滑肌细胞,神经性疾病：,神经原细胞 神经原,胶质细胞,间质,内皮细胞（高血压脑病）,9,间质细胞,血管内皮细胞,淋巴管内皮细胞,淋巴细胞,巨噬细胞,触突状细胞,Dendritic,cell,Cytokine (,细胞因子,),Cytokine Receptor,细胞因子受体,Chemokine,(,趋化因子,),Chemokine,Receptor,趋化因子受体,恶性肿瘤,除肿瘤细胞外，还有重要的细胞：,10,CANCER:,Multiple Cross-talk among Cancer,stromal,and endothelial Cells,Cancer cells,Stromal,cells,Endothelial,cells,Intercellular,Cross talk,?,Cancer,cells,Matrix (ECM),Endothelial cells,Pericytes,Stromal,cells,11,Stroma,:,1.,Fibroblasts in tumor:,“Activated fibroblast”,GF,GF R activated,2.,Genetic alteration:,Juvenile,polyposis,syndrome (JPS), Cowen syndrome etc,Dominant hereditary,Stromal,cells abnormality,Hamartoma,High risk for colon cancer,PTEN and others,“Landscaper”,“,Stroma,abnormal,microenviroment,”,“,Microenviroment,of tumor-host interface ”,12,ANGIOGENESIS,Two Processes,Endothelial Cells (EC),growth,migration,Vessel formation,Tube formation,extension,Other cells: Fibroblasts etc,13,ANGIOGENESIS / CANCER INVASION,EC Growth control & Vessel formation,Hypoxia,Activating hypoxia responses genes,( p53 ),Acidosis,VEGF-A,( Cancer. EC ),14,Protease / matrix / Cancer cell invasion / Angiogenesis,Proteases:,Degradation of ECM and Cell Associated Proteins,MMPs,(,Marix,metallo,proteinases,), membrane - anchored,MMP 2, MMP 9, (Cancer Cell),Soluble MMP (fibro, EC),Adamalysin, related membrane,proteinases,BMP 1,metalloproteinases,Tissue serine,proteinases,tPA,UK,Thrombin,Plasmin,uPA,( EC, fibro. ),uPAR,( Cancer ),Inhibitors,PAI,TIMPs,( Tissue inhibitor of,MMPs,),PEX ( Degradation product of,ECM by MMP 2 ),15,Proteases further create,Plasminogen,Angiostatin,(38KD) EC ATP,synthase,( I ),Procollagen,XXIII,Endostatin,(20KD),Prolaction,16KD,prolactin,ECM PEX EC,integrin,a,b3,( I ),RGD fragment,Cancer FAK (S),Troponin,Tn-1 EC ATP,Synthase,( I ),(Human cartilage),?,Inhibitors of Angiogenesis,Stimulators of Cancer Invasion,( I ),ANGIOGENESIS / CANCER INVASION,16,EC Receptors:,( EC and Cancer Cell ),VEGF R 1 (Flt-1),VEGF R 2 (Flk-1, KDR) VEGF A-D,PLGF,VEGF R 3 (Flt-4),VEGF C, D,TIE 1,?,TIE 2,Ang,1, 2,3? 4?,*,Integrin,a,b3,* ECM PEX,a,b5,(,Laminin,tenasin,),CD 36,Thrombospondin,(ECM Glycoprotein),cell adhesion, motility & growth,metallospondin,Vascular,Tubule,formation,*,Eph B1,Recognizing some,ligands,or partners,NHE 3,?,ARNT,?,EPA-1,?,Tenasoin,: new ECM.,ANGIOGENESIS / CANCER INVASION,17,VEGF,Rs,VEGF 121,VEGF 121,VEGF 145,VEGF 165,VEGF 165,VEGF 165 VEGF 165,VEGF B,VEGF C,VEGF D,VEGF C,VEGF D,PLGF 1,PLGF2,PLGF2,Semaphorin,III,(,Stmulation,of,phosphorylation,of VEG R2(KDR),VEGF R1 VEGF R2 VEGF R3,Neurophilin,1,Neurophilin,2,(flt-1) (flk-1 KDR) (,flt,4),Ligands,:,18,EC Growth Factors:,Specific,GFs,: VEGF A-D VEGF R2 (KDR. Flk-1),PLGF VEGF R1 (flt-1),Non-specific,GFs,(Local): Some of them:,autocrine,(EC, Cancer Fibro, ),TGF,a,EGF R,TGF,b,( fibro),TGF,b,R,( Cancer ) TGF,b,/ ECM TGF,b,released,aFGF,bFGF,FGF R,PDGF PDGF R,HGF Met,ANGIOGENESIS / CANCER INVASION,Protease,19,Chemokines,CXC ,chemokines,Recruit,monocytes,(Tumor cell origin) leukocytes,or others,CXC L12 CXC L-12 enriched tissue,CCR4 metastasis,Pseudopodia, migration, penetration of ECM, homing,Induce,TNFa,Cytokines related to EC or Capillary formation,IFN,a,Downregulate,VEGF production,bFGF,IFN,g,Inhibit angiogenesis,IL-12,Upregulate,IFN-,g,& IP-10, inhibiting angiogenesis,ANGIOGENESIS / CANCER INVASION,20,Interaction between cancer cells & EC,Hypoxia,Proteases Degradation of ECM,VEGFs,Angs,TGE,a,b,FGF,PDGF ?,Chemokines,cancer,EC,ANGIOGENESIS / CANCER INVASION,Metastasis,21,Enigmas,1.,Tumor / endothelial cells,Cross-talk,2.,Tumor cells,endothelia like cells.,Morphological evidence: yes,Biochemical: ?,3.,Stromal,cells / tumor cells,Stromal,cells / endothelial cells,4.,Genetic alteration of tumor vascular endothelial cells ?,Yes or not?,5.,Genetic alteration of tumor,stromal,cells,Cross-talk,22,APOPTOSIS,1. Death signals:,TNF,a,family,2. Death Receptors: TNF Receptor family, Decoy Receptor,3. Adaptor molecules: Interact with receptor,Caspase,(,Protocaspase,),4. Proto,caspases,: Activated,Caspases,5.,Effector,molecules: Other Proteases, DNA degradation enzymes,6. Inhibitors: For,Caspases,Adaptors,Others,New Progress,Molecules inside mitochondria, release into cytoplasm,(,Procaspases,cytochrome,C,ctc,.),Nuclear /,Cytoplamic,molecules ,translocate,to MT membrane,23,Mitochondrion / Cytoplasm,Caspases,Other,Signals,In,intermembrane,space,Protocaspase,2,9,3,Latent AIF,Cytochrcme,C,Smac,/ DIABLO,Life,Activation of,Protocaspase,3, 6, 7,Activation of,Protocaspase,9,Other,targest,Mitochondrion,Protocaspase-2,Protocaspase-3,Protocaspase-9,Apaf,1,Cytochrome,c,AIF,Others ? (,Smac,/ DIABLO),Life or Death ?,Alteration of Molecular,Compartmentation,(Mitochondrion versus Cytoplasm),Other Targets,Intermembrane,Space,AIF,(Apoptosis inducing factor) Science, 397: 3879 1999,Smac,(Second MT-derived activator of,caspase,),Or,DIABLO,(Direct IAP-binding protein at Low,pI,),Bcl,2,guard ?,Death,24,Mitochondria-death Signal Integrators,C. Bremer & G.,Koemer,(Science. Aug. 18.289: 1150, 2000),Translocate,to,mitochondrin,Stimulation / induction,Inhibition / block translocation,(Keep in,cytosol,),PTPC:,Permeability Transition Pore Complex (,Bax,etc.,),Bcl2 /,Bax,/ Bid,permeabilize,the outer MT membrane upon interaction with PTPC,Bid may be independent,JNK:,(SAPK, stress activating protein,kinase,) -, inactivate,Bcl,X,L,p53:,Translocated,to MT & interacted with,hsp,70,PKC:,Ttranslocated,to MT,P,25,Enigmas in Apoptosis,Translocation of nuclear or,cytosolic,protein molecules into mitochondria,Phosphorylation,/,dephosphorylation,Translocator,Protein / protein binding,conformational change,Binding to mitochondria surface,membane,protein,Translocation or release of proteins or other molecules from,mitochordria,to cytoplasm,Permeability transit pore complex,Voltage,depedent,anion channel (VDAC),Apoptosis induced by paradoxical signals,Growth factor / receptors,Protooncogenes,:,Ras,c-myc,etc.,26,HOT SPOTS OF SIGNALING,20% of 32,000 human coding gene: Signal transduction,Protein,Kinase,520,Protein,Phosphatase,130,Dominant,Oncogenes,100 PK,Tumor Suppressor,30 PK,Known PTK,PTK 90 (May, 2001),RPTK 58 (20 families),Non-receptor, PTK 32 ( 10 families),Cytoplasmic,27,Human RPTK,28,c-src,:,Truncated mutation of C to,Tyr,530,autophosphorylation,related to STAT-3,in colon cancer,c-abl,:,CS 9 (9:22),abl,/ BCR in ph1 CS,DNA damage induced apoptosis.,ATM,c-abl,bind,Rb,at G0/G1 (released after RB,phosph,. of,abl,prevent DNA-damage induced,abl,phosph,.,PDGF,Induce,cell,motility,& adhesion (,c-abl,involved in),BCR /,abl,Cyteplasm,X, nucleus,Human,Cytoplasmic,PTK,P,29,1.,Class I,p110 (catalytic),Adaptor / regulator,IA: RPTK activated,IB: hetero-,trimeric,G protein coupled Receptor activated.,Class II,p1(3)k,2.,Substrate:,ptd,Ins (4.5) P2,ptd,Ins (3.4.5) p3,Some P1(3k) (I & III):,+,Ser /,thr,K,3.,Isoform,a, b, d,of p110. Subunit,p85,a, p85,b, p55,g,of adaptor,4.,Binding of protein to lipid,IA,FVVE domain,ptd,Ins (3) P,PH(pleckstrin,homology) domain,PH(+) protein: 3-phospho-inositide dependent K (PDK 1) (ser/,thr,PK),Akt,(PKB),PI(3)K /,Akt,&,mTOR,/ p70,56k,(,rapamycin,target),PI(3k):,hetero,dimer,IA,ptd,Ins(3,4) P2,ptd,Ins(3,4,5)P3,30,Akt,( PKB ),Akt,:,v-,Akt,homolog,PKB,a, b, g,isoforms,N PH domain,Central,kinase,domain activation loop:,Thr,. 308,C Regulatory site: Ser 473,PDK- 1 (,3-phospho-inositide dependent,Kinase,1,),:,Thr,308,Akt,kinase,C PH domain,10 fold binding affinity to lipid membrane,(constitutively binding),PDK-2: Ser 473,Akt,31,Substrate of Akt-1,32,Substrate of,Akt,: 13: RXRXXS/T motif,(this motif:also for MAPKAPK-1 & p70,56K,),Class 1,Class 2,Anti-apoptosis,Regulator of cell growth,FKHR (,Forkhead,TA),(, ),eNOS,(, ),Bad,(, ),BRCA 1,(, ),CREB (,cAMP,Response element),(, ),GSK-3 (Glycogen,synthase,K),(, ),(,Cyclin,D,) (,b,-catenin, ),PDE-3B (,Phosphodiesterase,3B),(, ),p21WAF-1 (, via FKHR ),Two major class,33,RPTK,activation,P1(3)k,Ptd,Ins (3,4,5) P3,Ptd,Ins(3,4)P2,Interact with,AKT,Akt,/ PDK1 ,Akt,translocatin,to membrane,Thr,308,(loop), Ser 473,(Reg. Site),(PDK2 ?),PDK1/Akt activation:,Akt,P,P,translocate,to nuclei,AKT,P,P,PDK1,also, ,PKC,isoforms, serum and,glucoorticoid,induced K (GSK),PKC related K (PRK),p70,56k,p21 activated PK (PAK),P,34,PTEN,LOF of PTEN,Akt,also p27,LOF in,glioblastoma, germ cell cancer, breast cancer,Cancer & Pl (3)K IA /,Akt,Akt,expression, :,pancreatic, ovarian cancer,Akt, /,p65,(mutant of p85,a,Pl(3)k),p110 (Pl(3)K) amplification (ovarian cancer),Pl(3)K IB (p110 g ) LOF: colon cancer,(Bcl2, CDK.,Cyclin,D,),(3-phosphoinositide,phosphatase, TS),ptd,Ins (3,4,5) P3,ptd,(3,4) P2,35,mTOR,( Mammalian Target of,Rapamycin,),1. Conserved family:,Yeast: TOR1, TOR2, MEC1, TEL1,Rad,3,Drosophila: MEC41,Mammal:,mTOR, ATM, ATR ( ATM related ),TRAPP ( transformation / transcriptional domain-associated P ),2.,mTOR,= FK506 binding P (FKBP) (human),Rapamycin,associated P (FRAP human),Rapamycin,and FKBP12 target-1 (rat),3.,mTOR,C terminal K domain (homologous to,kinase,domain of Pl(3)K and Pl(4)K,ser / the K activity only,mTOR,c-myc, (inhibited by,mTOR,inhibitor,rapamycin,), stat 3 ( ser 727), PKC ; PKC , 4E-BP dissociation & release of elf-4E, cap dependent initiation, translation of 5-UTR,P,P,36,P1 (3) K dependent,cyelin,D3 ,Phosphorylation,of,Rb,and p107, E2F,Involved in P1 (3) K and,Akt,tumorigenesis,Dependent on,Phosphorylation,& activation of p70,S6K,Ribosomal S6,Kinase,S6K1, S6K2,Regulator of,cell growth,& protein translation,p70,S6K, short,isoform,of S6K 1,Cytoplasic,in large part,Activation is blocked by,rapamycin,Mediate,mTOR,effect on protein-translation,Phosphorylation,of S6 subunit (40s,rs,protein),S6, translation of 5,ter,.,Pyr,-rich tract (5-TOP mRNA),Also involved in cell cycle,37,Wnt,Pathway,Wnt,Wnt,1 = ( int-1, mouse MMTV integration activated gene),Porcupine: A gene for,wnt,secretion,Polytopic,membrane protein,Related to,acyl,transferase,Frizzled (,Fz,),Wnt,Receptor,Dally: Co-receptor of,Wnt,Glypican,-type,heparan,sulfate,proteoglycan,Glycosyl-Ptd,Ins moiety, membrane,b,-catenin,: in absence of,Wnt,Destabilized by a protein complex,Dishevelled,(,Dsh,) activated by,Wnt-Fz,binding,Axin,APC,GSK-3,b,(Glycogen,synthase,kinase,),GSK - 3b -,b,-catenin, -,b,catenin, degradation,Wnt,Fz,binding,Dsh,GSK - 3,b,b,-catenin,stabilized,enter nucleus,Nuclear transcription factor:,Tcf,/ LEF: activated by,b,-catenin,P,P,38,Wnt,Pathway,Dsh,APC,GSK 3,b,b,-catenin,Axin,TCF / LEF,APC,GSK 3,b,Dsh,Axin,TCF / LEF,b,-catenin,b,-catenin,Wnt,LRP,Fz,39,Hedgehog,Pothway,Hedgehog: Embryonic development inducer,Hh,hh,:,Humna,Shh,(Sonic,hh,),Ihh,(Indian,hh,),Dhh,(Desert,hh,),19KD protein C Cholesterol ester,N ,Palmitoylation,(after cholesterol addition, enhancing signaling),Secretion of,hh,:,2 genes:,Dispatched,related to,hh,R Patched,hh,release,Toutvelu,:,enzyme, heparin-SO,4,synthesis for transport and response of,hh,Receceptor,of,hh,Patched (,Ptc,):,Ts, 12 Span-,transmembrane,Proton-driven lipid,transportor,Smoothened (,Smo,):,Onc, 7-transmembrane p (,Fz,like ),40,1.,hh,(-): ( in a complex),hh,(+):,2.,Smo,activated:,coupled with a complex,3.,hh,(-):,(1),Ci,N,ter,. transcription repressor (,Ci,75),(2),Ci,in cytoplasm ( Cos2 / Fu / Su /,Ci,complex anchored on microtubule),4.,hh,(+): Costa / Fu / Su /,Ci,dissociated from microtubule,Ci,(Full length): enters nucleus,Transcription of genes (,ptc, Hip and growth genes),Activation of,hh,Pathway,Ptc,Smo,hh,Ptc,Smo,*,hh,-,Ptc,Smo,*,Smo,*,Fu,Cos,2,Su,Ci,Fu: Fused, S / T PK,Su: Suppressor of Fu,Costa 2 ( Cos. 2 ) :,Kinase,like,Ci,:,Cubitus,interuptus,ZF TA,Gli,in mammal,PKA,41,Su,Fused (Fu),Costa 2,PKA,Smoothened,(,Smo,),Patched,(,Prc,),Hedgehog (,Hh,),C i,C i,Growth,gene,product,Ptc,Hip,Patehed,:,Disrfunctin,in BCC,Smo,: mutated in breast Hedgehog Pathway,MiT,microtubule,Mit,Su,Fused (Fu),Costa 2,C i,(,Ci,75 ),Mit,42,43,