摘要
ErbB2是原癌基因erbB-2编码的185kDa的细胞膜受体,为表皮生长因子受体(epidermal growth factor receptor,EGFR)家族成员之一。该家族包括ErbB-1(又称EGFR,HER1)、ErbB2(又称HER2)、ErbB-3(HER3)和ErbB-4(HER4)四个成员。ErbB2高表达肿瘤细胞中Ras-MAPK和PI3K-Akt信号传导活性较高,细胞增殖能力较强,分化成熟和凋亡机制受到抑制,细胞恶性程度高。ErbB2高表达肿瘤细胞可抵抗TNF-α、射线以及各种化疗药物引起的细胞凋亡效应。临床上ErbB2表达与患者预后密切相关,ErbB2高表达的患者易发生肿瘤转移,存活期短。由于ErbB2在正常细胞和肿瘤细胞中的表达水平具有显著的差异,因而已成为肿瘤免疫生物治疗的理想靶点,亦是目前肿瘤治疗研究领域的热点分子。Herstatin是近年来发现的唯一天然存在的ErbB2自身抑制分子,它是ErbB2mRNA加工过程中选择性剪接的产物,是一种可溶性的ErbB2受体。Herstatin能与细胞表面的ErbB2受体高亲和力结合,阻碍ErbB2同源或异源二聚体的形成,抑制受体酪氨酸磷酸化及ErbB2过表达细胞的增殖能力。但是,Herstatin与ErbB2相互作用的机制及与ErbB2受体分子的结合位点以及抑制ErbB2过表达细胞增殖的机制均不清楚。阐明ErbB2分子表达的自身调节机制,有助于理解该受体分子在生理条件下表达平衡对于细胞正常分化的重要意义,而且可能为ErbB2过表达肿瘤的治疗提供新思路。对二者相互作用位点的分析亦可为寻找到控制ErbB2的敏感靶位及有效的小分子抑制剂的设计提供依据。
本研究采用基因工程技术构建了Herstatin的真核表达载体,将构建成功的载体转染ErbB2表达水平不同的细胞,在mRNA和蛋白水平均检测到Herstatin的表达。应用MTT法证实了Herstatin对细胞的增殖具有明显的抑制作用。Hoechst和PI染色发现Herstatin转染后可诱导转染细胞凋亡,这可能是其抑制细胞增殖
ErbB2, a 185 kDa receptor, is encoded by ErbB2 oncogene. It is one of the members of epidermal growth factor receptor (EGFR) family, which consists of four receptors including epidermal growth factor receptor (EGFR, also called HER-1 or ErbB-1), ErbB2 (HER-2), ErbB3 (HER-3) and ErbB4 (HER-4). ErbB2 overexpression is associated with poor progrnosis and short time to relapse. ErbB2 also enhances the metastatic potential of cancer cells. The most important intracellular signal transduction pathways activated by ErbB2 are Ras-MAPK and PI3K-Akt which play an important role in proliferation, differentiatio, even resistanse to TNF- α ,γ -radiation and chemotherapy. ErbB2 is frequently overexpressed in many human tumors. Therefore, ErbB2 is a good candidate target for immunotherapy and also a research hotspot. Recently, herstatin, an autoinhibitor of ErbB2, has been reported. It consists of subdomains L1 and S1 of ErbB2 and a novel C-terminal sequence encoded by intron 8. It has been demonstrated that Herstatin can bind to ErbB2 with high affinity. It disrupts the formation of heterodimers with EGFR or HER3 and homodimer with itself, reduces tyrosine phosphorlation of EGFR family and inhibits proliferation of cancer cells that overexpress ErbB2. However, the mechanism of the interaction between Herstatin and ErbB2 has not been fully understood and the binding sites are not clear now. The binding region of Herstatin on ErbB2 ectodomain might be a potential target region for the drug design.In the present study, the full-length sequence of Herstatin was amplified by over-lapping PCR. A plasmid containing Herstatin was constructed. The cells were transfected with the plasmid pcDNA3.1/Hst and Herstatin was expressed in the transfected cells. Herstatin mRNA and an about 60 KDa protein were detected by RT-PCR and West-blotting respectively. The results showed that Herstatin can inhibit cell proliferation and induce apoptosis by using Hoechst staining and flowcytometry.To investigate the interaction site on ErbB2 with Herstatin, the 3-D structure of Herstatin and ErbB2 complex was constructed by using computer-aided homology method. Based on the predicted secondary structure of C-terminal region of 79 aa by using GOR IV method, the 3-D model of the sequence of aa 341-419 representing C-terminal region of 79 aa was constructed by ab initio modeling method. The 3-D sequence of Herstatin from aa 1-340 were derived from the 3-D crystal structure of ErbB2 ECD. According to the 3-D crystal structure of ErbB2 ECD, the theoretical spatial structure of ErbB2 ECD was modeled with computer-aided homology method. Subsequently AUTODOCK3.0 program used to dock Herstatin- and ErbB2, the final optimized 3-D complex structure of ErbB2 ECD and Herstatin was modeled. The interaction region of ErbB2 ECD was at the C-terminal portion of S1, while the binding domain of Herstatin to ErbB2 ECD was at the 79 aa region encoded by intron 8.
引文
1. Schechter AL, Stem DF, Vaidyanathan L, et al. The neu oncogene: an erb-B-related gene encoding a 185,000-Mr tumour antigen. Nature, 1984, 312:513-516.
2. Bajaj M, Waterfield MD, Schlessinger J, et al. On the tertiary structure of the extracellular domains of the epidermal growth factor and insulin receptors. Biochim. Biophys. Acta, 1987,916:220-226.
3. Cho HS, Leahy DJ. Structure of the extracellular region of HER3 reveals an interdomain tether. Science, 2002, 297:1330-1333.
4. Garrett TP, McKern NM, Lou M, et al. Crystal structure of the truncated epidermal growth factor receptor ectodomain bound to transforming growth factor. Cell, 2002,110:763-773.
5. Harari D, Yarden Y. Molecular mechanisms underlying ErbB2/HER2 action in breast cancer. Oncogene, 2000,19(53): 6102-6114.
6. Garrett TP, McKern NM, Lou M, et al. The crystal structure of a truncated ErbB2 ectodomain reveals an active conformation, poised to interact with other ErbB receptors. Mol Cell, 2003, 11(2):495-505.
7. Coussens L, Yang-Feng TL, Liao YC, et al. Tyrosine kinase receptor with extensive homology to EGF receptor shares chromosomal location with neu oncogene. Science, 1985, 230:1132-1139.
8. Franklin MC, Carey KD, Vajdos FF, et al. Insights into ErbB signaling from the structure of the ErbB2-pertuzumab complex. Cancer Cell, 2004, 5(4):317-328.
9. Cho HS, Mason K, Ramyar K, et al. Structure of the extracellular region of HER2 alone and in complex with the Herceptin Fab. Nature, 2003, 421:756-760
10. Sundaresan S, Roberts PE, King KL, et al. Biological response to ErbB ligands in nontransformed cell lines correlates with a specific pattern of receptor expression. Endocrinology, 1998,139:4756-4764.
11. Klapper LN, Glathe S, Vaisman N, et al. The ErbB-2/HER2 oncoprotein of human carcinomas may function solely as a shared coreceptor for multiple stroma-derived growth factors. Proc Natl Acad Sci U S A, 1999,96(9):4995-5000
12. Kumagai T, Davis JG, Horie T, et al. The role of distinct p185neu extracellular subdomains fordimerization with the epidermal growth factor (EGF) receptor and EGF-mediated signaling. Proc. Natl. Acad. Sci. USA, 2001 98:5526-5531.
13. Marmor MD, Skaria KB, Yarden Y. Signal transduction and oncogenesis by ErbB/HER receptors. Int J Radiat Oncol Biol Phys, 2004, 58(3): 903-913
14. Lee RJ, Albanese C, Fu M, et al. Cyclin D1 is required for transformation by activated Neu and is induced through an E2F-dependent signaling pathway. Mol Cell Biol, 2000, 20(2): 672-83.
15. Zhou BP, Hu MC, Miller SA, et al. HER-2/neu blocks tumor necrosis factor-induced apoptosis via the Akt/NF-kappaB pathway. J Biol Chem, 2000, 275(11): 8027-31.
16. Soderlund K, Perez-Tenorio G, Stal O. Activation of the phosphatidylinositol 3-kinase/Akt pathway prevents radiation-induced apoptosis in breast cancer cells. Int J Oncol, 2005, 26(1): 25-32.
17. Knuefermann C, Lu Y, Liu B, et al. HER2/PI-3K/Akt activation leads to a multidrug resistance in human breast adenocarcinoma cells. Oncogene, 2003, 22(21):3205-3212
18. Downward J.PI 3-kinase, Akt and cell survival. Semin Cell Dev Biol, 2004, 15(2):177-82.
19. Casalini P, Botta L, Menard S. Role of p53 in HER2-induced proliferation or apoptosis. J Biol Chem, 2001,276(15): 12449-12453.
20. Mendelsohn J, Baselga J. The EGF receptor family astargets for cancer therapy. Oncogene, 2000, 19:6550-6565.
21. Lohrisch C, Piccart M. An overview of HER2. Semin Oncol. 2001,28(6 Suppl 18): 3-11.
22. Burstein HJ, Harris LN, Gelman R, et al. Preoperative therapy with trastuzumab and paclitaxel followed by sequential adjuvant doxorubicin/cyclophosphamide for HER2 overexpressing stage Ⅱ or Ⅲ breast cancer: a pilot study. J Clin Oncol, 2003, 21(1): 46-53.
23. Yan DH, Chang LS, Hung MC. Repressed expression of the HER-2/c-erbB-2 proto-oncogene by the adenovirus E1a gene products. Oncogene, 1991, 6(2):343-345.
24. Zhang L, Lau YK, Xia W, et al. Tyrosine kinase inhibitor emodin suppresses growth of HER-2/neu-overexpressing breast cancer cells in athymic mice and sensitizes these cells to the inhibitory effect of paclitaxel. Clin Cancer Res, 1999, 5(2):343-53.
25. Allen LF, Eiseman IA, Fry DW, et al. CI-1033, an irreversible pan-erbB receptor inhibitor and its potential application for the treatment of breast cancer. Semin Oncol, 2003, 30(5 Suppl 16): 65-78.
26. Berezov A, Chen J, Liu Q, et al. Disabling receptor ensembles with rationally designed interface peptidomimetics. J Biol Chem, 2002, 277(31):28330-28339
27. Allal C, Sixou S, Kravtzoff R, et al. Molecular BioVectors (SMBV) improve antisense inhibition of erbB-2 expression. Br J Cancer, 1998, 77(9): 1448-1453.
28. Berezov A, Greene MI, Murali R. Structure-based approaches to inhibition of erbB receptors with peptide mimetics, Immunol Res, 2003,27(2-3): 303-308.
29. Doherty JK, Bond C, Jardim A, et al: The HER2/neu receptor tyronsine kinase gene encodes a secreted autoinhibitor. Proc Natl Acad Sci.USA, 1999,96:10869-10874.
30. Azios NG, Romero FJ, Denton MC, et al. Expression of herstatin, an autoinhibitor of HER2/neu, inhibits transactivation of HER-3 by HER2 and blocks EGF activation of the EGF receptor. Oncogene, 2001, 20:5199-5209.
31. Alimandi M, Romano A, Curia MC, et al. Cooperative signaling of ErbB3 and ErbB2 in neoplastic transformation and human mammary carcinomas. Oncogene, 1995,10(9): 1813-1821.
32. Justman QA, Clinton GM. Herstatin an autoinhibitor of the human epidermal growth factor receptor 2 tyronsine kinase, modulates epidermal growth factor signaling pathways resulting in growth arrest. J Bio Chem, 2002, 277(23):