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顾 健 人

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. 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？. 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

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顾 健 人

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  1. 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

  2. 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？ 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

  3. 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

  4. 一个基因可编编码两种完全不同的蛋白质 p16 Ink 4a E1 b E1 a Exon 2 Exon 3 P14 ARF (human) P19 ARF (mouse) Cross Talk betweren Oncogenes & tumor suppressors Oncogenic Signals Ras, c-myc, E2F P14 ARF (Block MDM-2 / p53 binding) p16 Ink 4a DNA Damage p53 on Rb on Cell cycle arrest

  5. 功能基因组学Functional Genomics 在基因组的水平 • 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：

  6. 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。

  7. 细胞／分子细胞生物学／重要问题 生 死 细胞 凋亡 细胞 增殖 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, that’s a question” Hamlet－Shakespear

  8. 细胞 凋亡基因 Apoptotic gene DNA 复制基因 原癌 基因 Proto- oncogene 抑癌 基因 Cancer Suppressor DNA 转录 细胞周期 基因 M （抗凋亡） Antiapoptotic （促进凋亡） Check Points of Cell Cycle G1  S check point G2  M check point M: Mitotic Spindle checkpoint

  9. 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）

  10. 恶性肿瘤 除肿瘤细胞外，还有重要的细胞： 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

  11. CANCER: Multiple Cross-talk among Cancer, stromal and endothelial Cells Cancer cells Cancer cells Matrix (ECM) Endothelial cells Pericytes Stromal cells ? Stromal cells Endothelial cells Intercellular Cross talk

  12. 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 ”

  13. ANGIOGENESIS Two Processes Endothelial Cells (EC) growth migration Vessel formation Tube formation extension Other cells: Fibroblasts etc

  14. ANGIOGENESIS / CANCER INVASION EC Growth control & Vessel formation HypoxiaActivating hypoxia responses genes( p53 ) AcidosisVEGF-A ( Cancer. EC )

  15. 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 )

  16. ANGIOGENESIS / CANCER INVASION 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 fragmentCancer FAK (S) Troponin Tn-1 EC ATP Synthase ( I ) (Human cartilage) Inhibitors of Angiogenesis Stimulators of Cancer Invasion ? ( I )

  17. ANGIOGENESIS / CANCER INVASION 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 2Ang 1, 2 3? 4? Vascular Tubule formation * Integrin ab3 * ECM PEX ab5 (Laminin, tenasin) CD 36Thrombospondin (ECM Glycoprotein) cell adhesion, motility & growth metallospondin *Eph B1Recognizing some ligands or partners NHE 3? ARNT ? EPA-1? Tenasoin: new ECM.

  18. VEGF Rs VEGF R1 VEGF R2 VEGF R3 Neurophilin 1 Neurophilin 2 (flt-1) (flk-1 KDR) (flt 4) Ligands:

  19. ANGIOGENESIS / CANCER INVASION 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, ) TGFa EGF R TGFb( fibro) TGFb R ( Cancer ) TGF b / ECM TGF b released aFGF bFGF FGF R PDGF PDGF R HGF Met Protease

  20. ANGIOGENESIS / CANCER INVASION Cytokines related to EC or Capillary formation IFN a Downregulate VEGF production bFGF IFNg Inhibit angiogenesis IL-12 Upregulate IFN-g & IP-10, inhibiting angiogenesis 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

  21. ANGIOGENESIS / CANCER INVASION Interaction between cancer cells & EC Hypoxia Proteases Degradation of ECM VEGFs Angs TGEa bFGF PDGF ? Chemokines cancer EC Metastasis

  22. 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

  23. APOPTOSIS 1. Death signals: TNFa 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: ForCaspases Adaptors Others New Progress Molecules inside mitochondria  release into cytoplasm (Procaspases, cytochrome C ctc.) Nuclear / Cytoplamic molecules  translocate to MT membrane

  24. Activation of Protocaspase 3, 6, 7 Activation of Protocaspase 9 Mitochondrion / Cytoplasm Other targest Caspases Other Signals Bcl 2 guard ? Life In intermembrane space Protocaspase 2 9 3 Latent AIF Cytochrcme C Smac / DIABLO Intermembrane Space Mitochondrion Other Targets Protocaspase-2 Protocaspase-3 Protocaspase-9 Apaf 1 Cytochrome c AIF Others ?? (Smac / DIABLO) Death Life or Death ? Alteration of Molecular Compartmentation (Mitochondrion versus Cytoplasm) AIF(Apoptosis inducing factor) Science, 397: 387~9 1999 Smac(Second MT-derived activator of caspase) OrDIABLO(Direct IAP-binding protein at Low pI)

  25. P 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 XL p53:Translocated to MT & interacted with hsp 70 PKC:Ttranslocated to MT

  26. 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.

  27. HOT SPOTS OF SIGNALING 20% of 32,000 human coding gene: Signal transduction Protein Kinase520 Protein Phosphatase130 Dominant Oncogenes >100 PK Tumor Suppressor30 PK Known PTK PTK >90 (May, 2001) RPTK 58 (20 families) Non-receptor, PTK 32 ( 10 families) Cytoplasmic

  28. Human RPTK

  29. P Human Cytoplasmic PTK c-src:Truncated mutation of C’ to Tyr 530autophosphorylation related to STAT-3in 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

  30. PI(3)K / Akt & mTOR / p7056k (rapamycin target) PI(3k): 1.Class Ip110 (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.Isoforma, b, d of p110. Subunit p85a, p85b, p55g of adaptor 4.Binding of protein to lipidIA FVVE domain  ptd Ins (3) P PH(pleckstrin homology) domain PH(+) protein: 3’-phospho-inositide dependent K (PDK 1) (ser/thr PK) Akt (PKB) hetero dimer IA ptd Ins(3,4) P2 ptd Ins(3,4,5)P3

  31. 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

  32. Substrate of Akt-1

  33. Substrate of Akt: >13: RXRXXS/T motif(this motif:also for MAPKAPK-1 & p7056K) Two major class

  34. P P P P P PDK1/Akt activation: 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 ?) Akt AKT translocate to nuclei PDK1also－  PKC isoforms, serum and glucoorticoid induced K (GSK) PKC related K (PRK) p7056k p21 activated PK (PAK)

  35. PTEN (3’-phosphoinositide phosphatase, TS) ptd Ins (3,4,5) P3  ptd (3,4) P2 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  )

  36. P P 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

  37. Ribosomal S6 Kinase S6K1, S6K2 Regulator of cell growth & protein translation p70S6K – 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 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 p70S6K

  38. P P 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-3b (Glycogen synthase kinase) GSK - 3b -  b-catenin  - b – catenin  degradation Wnt – Fz binding  Dsh GSK - 3b b-catenin stabilized enter nucleus Nuclear transcription factor: Tcf / LEF: activated by b-catenin

  39. Axin Axin GSK 3b APC b-catenin Dsh Dsh b-catenin Wnt Wnt Pathway Fz LRP GSK 3b APC b-catenin TCF / LEF TCF / LEF

  40. 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:Dispatchedrelated to hh R Patched hh release Toutvelu:enzyme, heparin-SO4 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 )

  41. Ptc Smo Cos 2 Su Ci Fu Activation of hh Pathway 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) Fu: Fused, S / T PK Su: Suppressor of Fu Costa 2 ( Cos. 2 ) : Kinase like Ci: Cubitus interuptus, ZF TA Gli in mammal hh – Ptc Smo* hh - Ptc Smo* Smo* PKA

  42. Mit Costa 2 PKA Fused (Fu) C i ( Ci 75 ) Su Hedgehog (Hh) Patched (Prc) Smoothened (Smo) Mit Costa 2 Fused (Fu) C i MiT microtubule Su Ptc Hip C i Growth gene product Patehed: Disrfunctin in BCC Smo: mutated in breast Hedgehog Pathway

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