细胞信号转导与肿瘤 ——中山大学2005级《分子医学》博士生课程第四讲 2005年9月27日 细胞信号转导与肿瘤 ——中山大学2005级《分子医学》博士生课程第四讲 2005年9月27日 黎孟枫 医学博士 中山大学基础医学院微生物学教研室mengli@gzsums.edu.cn

引言：细胞信号转导与生命过程——问题的提出和理论的产生 细胞信号转导理论概述 信号转导研究中的重大理论问题及热点领域 信号转导的研究方法与工具 信号转导理论研究及应用举例：在肿瘤发生发展中的信号转导的意义 信号转导与肿瘤临床：诊断和治疗 细胞信号转导经典文献举例

引言 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……)

组织生长需要 细胞分裂增殖 细胞 生长因子 细胞周期 蛋白表达 病原体侵入 抗感染状态 细胞 抗原 细胞因子 表达分泌 细胞过度生长 细胞死亡 细胞 死亡因子 胞内致死 分子表达

抗凋亡因子 表达、激活 胞外信号 信号作用 于细胞 基因表 达改变 细胞表 型改变 细胞骨架蛋 白表达、激活 牵动细胞移动 (Cell movement) 趋化因子 细胞粘附 细胞存活 (Survival) 抗凋亡因子 表达、激活 胞外信号 信号作用 于细胞 基因表 达改变 细胞表 型改变

细胞信号转导理论概述

胞外信号分子(可溶性分子、细胞表面分子、组织基质分子) 靶细胞跨膜分子(狭义受体如EGFR或广义受体如Integrin) 靶细胞受体（胞内段）化学变化（如磷酸化、二聚体形成） 靶细胞内信号转导分子化学变化与激活 （如磷酸化、去磷酸化、聚体形成） 激活的信号转导分子进入胞核 进入胞核的转导分子作用于基因转录调控区基因表达改变

Extracellular Signal Molecules Growth Factors PDGF (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. Cytokines IFN- (Interferon- ), IFN-  (Interferon- ), TNF (Tumor Necrosis Factor), Interleukins (1, 2, 3, 4…) 3. Death molecules Fas 4. Adhesion molecules Cadherins, Adhesin… 5. Hormone Insulin 6. Stress

Signal Transducing Receptors Transmembrane receptors that have intrinsic enzymatic activity. ——Autophosphorylation ——Phosphorylation of other substrates A) Tyrosine kinases: PDGF-R, insulin-R, EGFR and FGF-R B) Tyrosine phosphatases: e.g. CD45 C) Guanylate cyclases: e.g. natriuretic peptide receptors) D) Serine/Threonine kinases: activin and TGF-b receptors

2. Receptors that are coupled, inside the cell, to GTP-binding and hydrolyzing proteins (G-proteins). e.g., adrenergic receptors, odorant receptors, and certain hormone receptors (e.g. glucagon, angiotensin, vasopressin and bradykinin). 3. Receptors that are found intracellularly and upon ligand binding migrate to the nucleus where the ligand-receptor complex directly affects gene transcription e.g., STAT1, 3, 4, 5, 6… (Signal transducer and activator of transcription ) 4. Simple receptors: e.g., ion-channels that lead to changes in membrane electric potential

信号转导过程中的生物化学 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 Tyrosine phosphorylation Receptor Tyrosine Kinases (RTKs) contains: An extracellular ligand binding domain. An intracellular tyrosine kinase domain. An intracellular regulatory domain. A transmembrane domain. Tyrosine phosphorylation Interact with and phosphorylate Src homology domain 2 (SH2)-containing proteins (e.g., PLC-, Ras, PI-3K, etc) Phosphorylate other kinases phosphorylate proteins, which upon phosphorylated, can enter the nuclear and bind DNA regulatory regions.

Characteristics of the Common Classes of RTKs Examples Structural Features of Class I EGF receptor, NEU/HER2, HER3 cysteine-rich sequences II insulin receptor, IGF-1 receptor cysteine-rich sequences; characterized by disulfide-linked heterotetramers III PDGF receptors, c-Kit contain 5 immunoglobulin-like domains; contain the kinase insert IV FGF receptors contain 3 immunoglobulin-like domains as well as the kinase insert; acidic domain V vascular endothelial growth factor (VEGF) receptor contain 7 immunoglobulin-like domains as well as the kinase insert domain VI hepatocyte growth factor (HGF) and scatter factor (SC) receptors heterodimeric like the class II receptors except that one of the two protein subunits is completely extracellular. The HGF receptor is a proto-oncogene that was originally identified as the Met oncogene VII neurotrophin receptor family (trkA, trkB, trkC) and NGF receptor contain no or few cysteine-rich domains; NGFR has leucine rich domain

Non-Receptor Protein Tyrosine Kinases (PTKs) Two non-receptor PTK families: 1) The archetypapl PTK familty: Src-related proteins 2) 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) Typical 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-Myc and Smad Ligands bind to the type II receptors Complexed with type I receptors Type II R phosphorylates type I receptor Initiation of signaling cascade

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 Receptors 1. >1000 GPCRs, most of which are orphan receptors) 2. Three 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

Intracellular Hormone 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 in Signal Transduction 1. Transmembrane PTPs: e.g., CD45. 2. Intracellular PTPs.

胞外信号分子(可溶性分子、细胞表面分子、组织基质分子) 靶细胞跨膜分子(狭义受体如EGFR或广义受体如Integrin) 靶细胞受体（胞内段）化学变化（如磷酸化、二聚体形成） 靶细胞内信号转导分子化学变化与激活 （如磷酸化、去磷酸化、聚体形成） 激活的信号转导分子进入胞核 进入胞核的转导分子作用于基因转录调控区基因表达改变

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 expressed 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?

替代通路 （Alternative Pathways)

信号转导的研究方法与工具

一、蛋白质磷酸化状态的检测 二、信号转导分子过度表达或过度激活 三、基因转录活性测定 1、免疫印迹 (phospho-protein specific antibodies) 2、免疫沉淀 (protein-specific antibody + phospho-AA antibody 3、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 transduction 2、Constitutively activated mutants 三、基因转录活性测定 1、Electrophoretic mobility shift analysis (EMSA) ２、Reporter gene expression detection

四、信号转导分子的表达或活性抑制 1、Anti-sense 2、RNAi 3、Gene knock-out 4、Dominant negative mutants Ligand-binding site Phosphorylation site Docking site Protein-protein binding site DNA binding site 5、Small-molecule inhibitors：e.g., tyrosine kinase inhibitor (TKi) 6、Inhibitory oligopeptides

信号转导在肿瘤发生发展中的意义

Signaling molecules involved in cancer development/progression Receptors Growth factor receptors: EGFR Hormone receptor: ER, AR Angiogenic receptros: VEGF, PDGF, IGF Death receptors The Integrin system

Transcription factors Transducers Ras Raf Rho family PI-3K/Akt Death transducers STAT-3 …… Transcription factors c-Myc c-Jun and c-fos

Biological Effects of Signaling Related to Cancer Development/Progression Cell immobilization Abrogation of apoptosis Activation of cell cycle and removal of cell cycle checkpoints Angiogenesis Cell invasion Metastasis Drug 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 activation by FKHR-L1 Akt  Bad  14-3-3 binding  Release of Bcl-2 and Bcl-X  Cell survival Akt GSK3 GSK3 catalytic activity turned off  Permitting activation of c-Myc and cyclin D PDK1  phosphorylation of other kinases (p70 S6-kinasse, CISK, PKC)  Cell growth and survival

信号转导与肿瘤临床 ——诊断、预防与治疗 Expression level, mutations and antibodies of signaling molecules in cancer diagnosis EGFR: lung, H&N, glioma… TGF- receptor (type II): lung, H&N, Ras Androgen receptor and downstream molecules Estrogen receptor and downstream molecules ……

Protein phosphorylation and cancer diagnosis Determination of single phosphorylated signaling molecules: EGFR… Phospho-protein profiling: proteomics Phospho-protein based imaging technology Signaling inhibitor and cancer prevention/ therapy EGFR-selective TKi: Iressa EGFR antibodies Farnesylation or Geranylgeranylation inhibitors targeting Ras and Rho VEGFR antibody (and TKi): Avastin

经典文献举例

Cell Volume 98, Issue 3 , 295-303 Stat3 as an Oncogene Jacqueline F. Bromberg1, Melissa H. Wrzeszczynska1, Geeta Devgan1, Yanxiang Zhao2, Richard G. Pestell3, Chris Albanese3 and James E. Darnell, Jr.    ,   , 1, § 1 Laboratory of Molecular Cell Biology, The Rockefeller University, New York, New York 10021-6399, USA 2 Laboratory of Molecular Biophysics, The Rockefeller University, New York, New York 10021-6399, USA 3 The Albert Einstein Cancer Center, Department of Developmental and Molecular Biology, and Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461, USA Received 30 March 1999;  Revised 23 June 1999.  Available online 27 September 2000

信号转导理论在各生命科学领域中的普遍意义 以本课程中各讲为例： 干细胞 蛋白质组学 细胞凋亡 肿瘤转移 血管增生