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1、细胞信号转导与肿瘤,一、信号转导与生命过程问题的提出和理论的产生,细胞信号转导理论建立以前的细胞生物学,细胞的显微结构（胞膜、胞浆、胞核）细胞的生理功能（生存、“活性”、分裂增殖、胞间连接、吞饮、分泌、迁移、死亡)细胞组分的生物化学（脂、糖、核酸、蛋白）细胞的超微结构和亚细胞结构（脂质双层膜结构、细胞器),二、细胞信号转导理论概述,信号传导的基本概念：细胞外因子通过受体(膜受体或核受体)结合引发细胞内的一系列生物化学反应，直至细胞生理反应所需基因表达开始的过程。即：信号从细胞外，通过膜到细胞核的过程。,Extracellular Signal Molecules,Growth FactorsP
2、DGF(Platelet-Derived Growth Factor),EGF(Epidermal Growth Factor),TGF-(Transforming Growth Factor-),EPO(Erythropoietin),NGF(Nerve Growth Factor),IGF(Insulin-like Growth Factor),TPO(Thrombopoietin)2.CytokinesIFN-(Interferon-),IFN-(Interferon-),TNF(Tumor Necrosis Factor),Interleukins(1,2,3,4)3.Death mo
3、leculesFas 4.Adhesion moleculesCadherins,Adhesin5.HormoneInsulin6.Stress,1、生长因子（growth factor）这是一大类种类繁多，以刺激细胞生长为其特征的多肽，因生理作用而命名。共同特点：（1）其受体：具有酪氨酸激酶活性（2）特异性：生长因子与相应受体结合（3）多样性：激活多种不同的传导通路（4）家族性：结构相似，生理功能相近（5)交叉性：部分生长因子能与二种以上的不同受体结合，这在同一家族的成员中更为多见。,2、细胞因子（cytokine）最主要的家族有白介素、白细胞刺激因子、干扰素等。对于刺激细胞生长，虽没有生长
4、因子那样重要，但对于特异的细胞（如淋巴瘤等），它们的作用（如白介素）对于细胞生长、活化是极其重要的。主要特点：它们的受体本身都不具有激酶活性。,3、粘附分子（adhesion molecules）细胞与细胞，细胞与基质的相互粘附作用不仅是胚胎发育所必需，而且在炎症、伤口愈合以及免疫反应等过程中发挥重要作用。细胞粘附的信号传导过程引起人们重视，是由于它们在肿瘤侵袭、转移中的非同寻常的作用。,4、激素、神经递质等 最显著的共同特点：通过G蛋白联接受体传递信号。这是目前所知种类最多的一类可以刺激细胞生长的细胞外因子，种类繁多，结构各异，分子大小相差悬殊，人们熟知的代表有生长激素、乙酰胆碱、肾上腺素等
5、。,Signal Transducing Receptors,Transmembrane receptors that have intrinsic enzymatic activity.AutophosphorylationPhosphorylation of other substratesA)Tyrosine kinases:PDGF-R,insulin-R,EGFR and FGF-RB)Tyrosine phosphatases:e.g.CD45C)Guanylate cyclases:e.g.natriuretic peptide receptors)D)Serine/Threon
6、ine kinases:activin and TGF-b receptors,酪氨酸激酶受体 大多数细胞生长因子的受体都含有酪氨酸激酶的肽链序列，这类受体统称酪氨酸激酶受体。这些受体具有极为相似的结构。细胞外的一段糖基化肽链是与配体（ligand）结合的部位；中间是单一的疏水性的跨膜区；然后是具有酪氨酸激酶活性的膜内区。根据肽链序列的相似性和其他一些结构上的特点，这些受体被分成若干家族,酪氨酸激酶受体结构图,糖基化肽链,膜内区,细胞膜,胞浆,胞外,具激酶活性,2.Receptors that are coupled,inside the cell,to GTP-binding and hyd
7、rolyzing proteins(G-proteins).e.g.,adrenergic receptors,odorant receptors,and certain hormone receptors(e.g.glucagon,angiotensin,vasopressin and bradykinin).,G蛋白联接受体G蛋白联接受体包括：大多数激素、神经多肽、神经递质的受体。与酪氨酸激酶受体区别：G蛋白联接受体有7个跨膜区,细胞膜,核膜,第二信使cAMP、cGMP、DAG、PIP3,G 蛋白连接受体,配体,3.Receptors that are found intracellula
8、rly and upon ligand binding migrate to the nucleus where the ligand-receptor complex directly affects gene transcription（胞内受体）e.g.,STAT1,3,4,5,6(Signal transducer and activator of transcription)4.Simple receptors:（离子通道受体）e.g.,ion-channels that lead to changes in membrane electric potential,信号转导过程中的生
9、物化学,磷酸化反应（酪氨酸激酶、丝/苏氨酸激酶）蛋白质构象改变去磷酸化反应（磷酸酶）受体或其他信号转导分子的聚体化,Signal Transducers,Receptor Tyrosine Kinases(RTKs)contains:An extracellular ligand binding domain.An intracellular tyrosine kinase domain.An intracellular regulatory domain.A transmembrane domain.,Non-Receptor Protein Tyrosine Kinases(PTKs)Tw
10、o non-receptor PTK families:1)The archetypapl PTK familty:Src-related proteins2)Janus kinase(Jak)family,Most non-receptor PTKs couple to cellular receptors that lack enzymatic activity themselves(e.g.,CD4,CD8,TCR and all cytokine receptors such as IL-2R,Receptor Serine/Threonine Kinases(RSTKs),Typic
11、al example:Receptors for the TGF-superfamily of ligands The TGF-superfamily include 30 multifunctional proteins,e.g.,activins,inhibins and the bone morphogenetic proteins(BMPs).17 RSTKs isolated are in 2 subfamilies:type I and type II receptors.Nuclear proteins responding to TGF-activation include c
12、-Myc and Smad,Non-Receptor Serine/Threonine Kinases 1)cAMP-dependent protein kinase(PKA)2)Protein kinase C(PKC)3)Mitogen activated protein kinases(MAPK or ERK)(requiring phosphorylation of both tyrosine and threonine)G-Protein Coupled Receptors1.1000 GPCRs,most of which are orphan receptors)2.Three
13、different classes of GPCR:GPCRs that modulate adenylate cyclase activity and produce cAMP GPCRs that activate PLC-g leading to hydrolysis of polyphosphoinositides:angiotensin,bradykinin and vasopressin receptors.Photoreceptor,第二信使,PKA,Ras,Raf,MEK,MAPK,配体,G蛋白连接受体,细胞膜,核膜,G蛋白连接受体通路,Intracellular Hormon
14、e Receptors 1.Residing within the cytoplasm.,2.The steroid/thyroid hormone receptor superfamily(e.g.glucocorticoid,vitamin D,retinoic acid and thyroid hormone receptors):bind steroid/thyroid hormone,translocate to nuclear and bind specific DNA sequences hormone response elements(HREs).,*Phosphatases
15、 in Signal Transduction 1.Transmembrane PTPs:e.g.,CD45.2.Intracellular PTPs.,三、信号转导研究中的重大理论问题及热点领域,信号转导通路的调控,磷酸化去磷酸化调控信号转导分子消长的调控（分子半衰期）不同通路之间的效应调控胞内内源性抑制物的调控功能,Cross-Talk,信号转导效应的特异性When and Where?,Cooperation with other signaling pathways?Pre-existing transcription co-factors differentially express
16、ed and activated in different cell types?Pre-existing co-activators of target proteins?Subcellular localization of transducers?Optimal level(or a threshold)of phosphorylation/dephosphorylation?,胞内激酶 蛋白激酶调节功能称之为“生命开关”：在整个基因组中，大约有的基因是编码蛋白激酶的，在细胞内，是这些蛋白激酶调节着数以万计的其它蛋白质的功能。胞浆内的激酶大多是丝氨酸苏氨酸激酶，与细胞生长关系最密切。近年
17、来研究最多的是MAP激酶通路（mitogen-activated protein kinase pathway,MAPK pathway）促细胞分裂剂激活性蛋白激酶,（1）MAPK的特性和生物功能 MAPK是丝氨酸苏氨酸激酶。它需要被一类双向特异性激酶所激活，即MAPK本身需要丝氨酸苏氨酸和酪氨酸同时磷酸化，才具有100的活性。激活MAPK的激酶是MEK（促细胞分裂激活性蛋白激酶），即MAPK/Erk kinase.,MAP激酶:是MAP激酶传导通路中的重要中继站和枢纽，平时位于胞浆内，一旦被激活，迅速运到细胞核内；或直接激活转录因子；或激活另外一些蛋白激酶，启动或关闭一些特定的转录，对刺激信
18、号作出必要的反应，调节细胞的正常生命活动。MAPK传导通路粗犷模式是：生长因子受体小G蛋白启动MAPK转录因子生物效应。最好的例子为，EGF-EGFR-Ras-c-Raf-MEK-MAPK-TCF(T细胞特异因子)-细胞生长,（2）MAPK的分类 现已知至少有个亚家族，但研究得比较详尽的有三个，即Erk、JNT/SAPK 和P38(HOG-酿酒酵母基因，编码特异性核酸内切酶)。每个亚家族都可能有多个成员。Erk激酶与细胞增殖有关；JNK激活与细胞应激及细胞凋亡有关；P38激活与炎症反应有关,四、信号转导的研究方法与工具,（一）蛋白质磷酸化状态的检测1、免疫印迹(phospho-protein
19、specific antibodies)2、免疫沉淀(protein-specific antibody+phospho-AA antibody3、流式细胞仪分析4、Luminex分析（二）信号转导分子过度表达或过度激活1、Overexpression by gene transduction2、Constitutively activated mutants,（三）基因转录活性测定1、Electrophoretic mobility shift analysis(EMSA)凝胶滞留法 检测特异的DNA序列和蛋白结合、Reporter gene expression detection,（四）
20、信号转导分子的表达或活性抑制1、Anti-sense2、RNAi3、Gene knock-out4、Dominant negative mutantsLigand-binding sitePhosphorylation siteDocking siteProtein-protein binding siteDNA binding site5、Small-molecule inhibitors：e.g.,tyrosine kinase inhibitor(TKi)6、Inhibitory oligopeptides,（五）激酶检测法（Kinase Assay）直接检测激酶的活性（六）Two-hy
21、brid方法 利用酵母细胞在体内检测蛋白-蛋白相互作用的方法,1.细胞内分布分析,Inhibit AR translocation by herb extracts,Mib(1nM)-+KMKKT(g/ml)-50 100 200 N C N C N C N C N C,ARTubulinPARP,N：细胞核C：细胞浆,AR was mostly localized in cytosol in the absence of androgen.Stimulation with R1881,AR translocated into the nucleus.Treated with R1881/em
22、odin,AR was mostly retained in the cytosol.,2.荧光标记,3.基因敲除,促红细胞生成素信号途径相关因素分析,五、信号转导在肿瘤发生发展中的意义,Signaling molecules involved in cancer development/progression,ReceptorsGrowth factor receptors:EGFRHormone receptor:ER,ARAngiogenic receptros:VEGF,PDGF,IGFDeath receptorsThe Integrin system,Transducers Ras
23、 Raf Rho family PI-3K/AktDeath transducersSTAT-3Transcription factorsc-Mycc-Jun and c-fosSTAT-3,在肿瘤的发生发展过程中，由于各种基因水平的变化，使细胞在信号传导网络上也表现出与正常细胞的许多区别，有一些通路处于异常活跃状态，有一些则传递受阻，可概括如下：、增殖失控 一些与细胞生长、分裂和增殖有关的信号传导通路处于异常活化状态。,（）生长因子（）生长因子受体包括（）蛋白激酶（）细胞周期调控因子（5）G蛋白2、凋亡受阻 在肿瘤细胞中，本应处于活化状态的一些信号通路，如DNA破坏所激活的凋亡通路往往传递信
24、号受阻。这类通路包括：（1）NF family(2)Fas/Fas ligand(3)Bcl-2/Bax(4)P53,c-Myc等,、侵袭与转移 肿瘤的侵袭与转移实际上是建立在细胞与细胞间信号传递异常的基础上，这些通路包括：(1)integrin传导通路(2)E-Cadherin(3)nm23-H1,H2 KAI-1/CD82(4)VEGF传导通路(5)基质金属蛋白酶（matrix metolloprototeinase,MMP),Biological Effects of Signaling Related to Cancer Development/Progression,Cell imm
25、obilizationAbrogation of apoptosisActivation of cell cycle and removal of cell cycle checkpointsAngiogenesisCell invasionMetastasisDrug resistance,Phosphorylation targets of PI-3K,Akt Forkhead-related transcription factor 1(FKHR-L1)14-3-3 binding FKHR-L1 retaining in cytosol abrogation of gene activ
26、ation by FKHR-L1Akt Bad 14-3-3binding Release of Bcl-2 and Bcl-X Cell survivalAkt GSK3 GSK3 catalytic activity turned off Permitting activation of c-Myc and cyclin DPDK1 phosphorylation of other kinases(p70 S6-kinasse,CISK,PKC)Cell growth and survival,六、信号转导与肿瘤临床诊断、预防与治疗,Expression level,mutations a
27、nd antibodies of signaling molecules in cancer diagnosisEGFR:lung,H&N,gliomaTGF-receptor(type II):lung,H&N,RasAndrogen receptor and downstream moleculesEstrogen receptor and downstream molecules,Protein phosphorylation and cancer diagnosisDetermination of single phosphorylated signaling molecules:EG
28、FRPhospho-protein profiling:proteomicsPhospho-protein based imaging technologySignaling inhibitor and cancer prevention/therapyEGFR-selective TKi:IressaEGFR antibodiesFarnesylation or Geranylgeranylation inhibitors targeting Ras and RhoVEGFR antibody(and TKi):Avastin,Anti-androgen/AR effects,PSA,The
29、 Virtual Prostate.GWU,Part A Introduction of AR,Androgen receptor(AR)belongs to the superfamily of nuclear hormone receptors,www.biograf.ch/projects.htmlbiograf.ch/projects.html,It shows a similarity to progesterone and glucocorticoid receptors.,AR has 919 amino acid residues.,Many proteins interact
30、 with AR,The basic functions of AR:1.Testosterone enters the cell and is reduced to 5a-dihydrotestosterone(DHT)by 5a-reductase.2.DHT binds to the androgen receptor,and the complex moves into the nucleus 3.Control transcription of androgen-dependent genes,Prostate cancer depends on AR to mediate the
31、effect of androgen on tumor initiation and progression.,Debes JD,and.Tindall DJ.N Engl J Med 2004;351(15):1488-1490,for his discoveries concerning hormonal treatment of prostatic cancer”,The Nobel Prize in Physiology or Medicine 1966,Charles B.Huggins,Part B How to deplete AR?,Suppress AR gene expre
32、ssionInhibit AR translocationIncrease AR break down-,LNCaP androgen/AR response markers,Dose-response(48 h),KMKKT,g/ml,Time course(50 g/ml),Hours of treatment,Apoptosis at 100 g/ml(72 h),Cell cycle G1 arrest(48 h),Colony formation in soft agar(14 days),KMKKT blocked androgen action,KMKKT inhibited a
33、ndrogen-stimulated cell proliferation,Mibolerone,nM,KMKKT inhibited androgen signaling to PSA(8 days),Mibolerone,nM,KMKKT suppressed PSA mRNA,KMKKT decreased AR abundance and blocked AR nuclear translocation,24 h 2 h,Effect of decursin on PSA,48 h,Decursin,g/ml,Decursin recapitulatedAGN and KMKKT actions,RT-PCR,IB,Decursin blocked AR nuclear translocation and PSA transcription,Decursin effect persisted after removal,Distinction from bicalutamide/Casodex,Anti-androgen/AR mechanisms,Effect of KMKKT on final tumor weight,Mean+SEM,N=910 mice.,P=0.017,P=0.007,Mean+/-SD,N=910 mice.,
