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標題: 表皮生長因子受體蛋白經由活化Cdk5/p35進而調控非小型細胞肺癌之細胞生長與移動
EGFR Modulates Non-Small Cell Lung Cancer Cell Proliferation and Migration through Cdk5/p35 Activation
作者: 洪慶宗
Hong, Ching-Tsung
關鍵字: non-small cell lung cancer;非小型細胞肺癌;EGFR;Cdk5/p35;表皮生長因子受體蛋白;Cdk5/p35
出版社: 生命科學系所
引用: 1. Jemal A, Siegel R, Xu J, Ward E: Cancer statistics, 2010. CA Cancer J Clin 2010, 60(5):277-300. 2. Rusch V, Klimstra D, Venkatraman E, Pisters PW, Langenfeld J, Dmitrovsky E: Overexpression of the epidermal growth factor receptor and its ligand transforming growth factor alpha is frequent in resectable non-small cell lung cancer but does not predict tumor progression. Clin Cancer Res 1997, 3(4):515-522. 3. Sun S, Schiller JH, Gazdar AF: Lung cancer in never smokers--a different disease. Nat Rev Cancer 2007, 7(10):778-790. 4. Olayioye MA, Neve RM, Lane HA, Hynes NE: The ErbB signaling network: receptor heterodimerization in development and cancer. EMBO J 2000, 19(13):3159-3167. 5. Cataldo VD, Gibbons DL, Perez-Soler R, Quintas-Cardama A: Treatment of non-small-cell lung cancer with erlotinib or gefitinib. N Engl J Med 2011, 364(10):947-955. 6. Jorissen RN, Walker F, Pouliot N, Garrett TP, Ward CW, Burgess AW: Epidermal growth factor receptor: mechanisms of activation and signalling. Exp Cell Res 2003, 284(1):31-53. 7. West KA, Linnoila IR, Belinsky SA, Harris CC, Dennis PA: Tobacco carcinogen-induced cellular transformation increases activation of the phosphatidylinositol 3''-kinase/Akt pathway in vitro and in vivo. Cancer Res 2004, 64(2):446-451. 8. Herbst RS, Heymach JV, Lippman SM: Lung cancer. N Engl J Med 2008, 359(13):1367-1380. 9. Calo V, Migliavacca M, Bazan V, Macaluso M, Buscemi M, Gebbia N, Russo A: STAT proteins: from normal control of cellular events to tumorigenesis. J Cell Physiol 2003, 197(2):157-168. 10. Malumbres M, Pellicer A: RAS pathways to cell cycle control and cell transformation. Front Biosci 1998, 3:d887-912. 11. Sears RC, Nevins JR: Signaling networks that link cell proliferation and cell fate. J Biol Chem 2002, 277(14):11617-11620. 12. Martino A, Holmes JHt, Lord JD, Moon JJ, Nelson BH: Stat5 and Sp1 regulate transcription of the cyclin D2 gene in response to IL-2. J Immunol 2001, 166(3):1723-1729. 13. Dhavan R, Tsai LH: A decade of CDK5. Nat Rev Mol Cell Biol 2001, 2(10):749-759. 14. Lew J, Beaudette K, Litwin CM, Wang JH: Purification and characterization of a novel proline-directed protein kinase from bovine brain. J Biol Chem 1992, 267(19):13383-13390. 15. Malumbres M, Harlow E, Hunt T, Hunter T, Lahti JM, Manning G, Morgan DO, Tsai LH, Wolgemuth DJ: Cyclin-dependent kinases: a family portrait. Nat Cell Biol 2009, 11(11):1275-1276. 16. Selkoe DJ: Translating cell biology into therapeutic advances in Alzheimer''s disease. Nature 1999, 399(6738 Suppl):A23-31. 17. Ohshima T, Gilmore EC, Longenecker G, Jacobowitz DM, Brady RO, Herrup K, Kulkarni AB: Migration defects of cdk5(-/-) neurons in the developing cerebellum is cell autonomous. J Neurosci 1999, 19(14):6017-6026. 18. Gilmore EC, Ohshima T, Goffinet AM, Kulkarni AB, Herrup K: Cyclin-dependent kinase 5-deficient mice demonstrate novel developmental arrest in cerebral cortex. J Neurosci 1998, 18(16):6370-6377. 19. Ohshima T, Ward JM, Huh CG, Longenecker G, Veeranna, Pant HC, Brady RO, Martin LJ, Kulkarni AB: Targeted disruption of the cyclin-dependent kinase 5 gene results in abnormal corticogenesis, neuronal pathology and perinatal death. Proc Natl Acad Sci U S A 1996, 93(20):11173-11178. 20. Fu AK, Fu WY, Cheung J, Tsim KW, Ip FC, Wang JH, Ip NY: Cdk5 is involved in neuregulin-induced AChR expression at the neuromuscular junction. Nat Neurosci 2001, 4(4):374-381. 21. Rosales JL, Lee KY: Extraneuronal roles of cyclin-dependent kinase 5. Bioessays 2006, 28(10):1023-1034. 22. Upadhyay AK, Ajay AK, Singh S, Bhat MK: Cell cycle regulatory protein 5 (Cdk5) is a novel downstream target of ERK in carboplatin induced death of breast cancer cells. Curr Cancer Drug Targets 2008, 8(8):741-752. 23. Goodyear S, Sharma MC: Roscovitine regulates invasive breast cancer cell (MDA-MB231) proliferation and survival through cell cycle regulatory protein cdk5. Exp Mol Pathol 2007, 82(1):25-32. 24. Kuo HS, Hsu FN, Chiang MC, You SC, Chen MC, Lo MJ, Lin H: The role of Cdk5 in retinoic acid-induced apoptosis of cervical cancer cell line. Chin J Physiol 2009, 52(1):23-30. 25. Strock CJ, Park JI, Nakakura EK, Bova GS, Isaacs JT, Ball DW, Nelkin BD: Cyclin-dependent kinase 5 activity controls cell motility and metastatic potential of prostate cancer cells. Cancer Res 2006, 66(15):7509-7515. 26. Lin H, Juang JL, Wang PS: Involvement of Cdk5/p25 in digoxin-triggered prostate cancer cell apoptosis. J Biol Chem 2004, 279(28):29302-29307. 27. Selvendiran K, Koga H, Ueno T, Yoshida T, Maeyama M, Torimura T, Yano H, Kojiro M, Sata M: Luteolin promotes degradation in signal transducer and activator of transcription 3 in human hepatoma cells: an implication for the antitumor potential of flavonoids. Cancer Res 2006, 66(9):4826-4834. 28. Chen F, Wang Q, Wang X, Studzinski GP: Up-regulation of Egr1 by 1,25-dihydroxyvitamin D3 contributes to increased expression of p35 activator of cyclin-dependent kinase 5 and consequent onset of the terminal phase of HL60 cell differentiation. Cancer Res 2004, 64(15):5425-5433. 29. Kim E, Chen F, Wang CC, Harrison LE: CDK5 is a novel regulatory protein in PPARgamma ligand-induced antiproliferation. Int J Oncol 2006, 28(1):191-194. 30. Lin H, Chen MC, Chiu CY, Song YM, Lin SY: Cdk5 regulates STAT3 activation and cell proliferation in medullary thyroid carcinoma cells. J Biol Chem 2007, 282(5):2776-2784. 31. Liu JL, Wang XY, Huang BX, Zhu F, Zhang RG, Wu G: Expression of CDK5/p35 in resected patients with non-small cell lung cancer: relation to prognosis. Med Oncol 2010. 32. Choi HS, Lee Y, Park KH, Sung JS, Lee JE, Shin ES, Ryu JS, Kim YH: Single-nucleotide polymorphisms in the promoter of the CDK5 gene and lung cancer risk in a Korean population. J Hum Genet 2009, 54(5):298-303. 33. Weishaupt JH, Neusch C, Bahr M: Cyclin-dependent kinase 5 (CDK5) and neuronal cell death. Cell Tissue Res 2003, 312(1):1-8. 34. Harada T, Morooka T, Ogawa S, Nishida E: ERK induces p35, a neuron-specific activator of Cdk5, through induction of Egr1. Nat Cell Biol 2001, 3(5):453-459. 35. Lee JH, Kim KT: Induction of cyclin-dependent kinase 5 and its activator p35 through the extracellular-signal-regulated kinase and protein kinase A pathways during retinoic-acid mediated neuronal differentiation in human neuroblastoma SK-N-BE(2)C cells. J Neurochem 2004, 91(3):634-647. 36. Lin H, Lin TY, Juang JL: Abl deregulates Cdk5 kinase activity and subcellular localization in Drosophila neurodegeneration. Cell Death Differ 2007, 14(3):607-615. 37. Lockwood WW, Chari R, Coe BP, Girard L, Macaulay C, Lam S, Gazdar AF, Minna JD, Lam WL: DNA amplification is a ubiquitous mechanism of oncogene activation in lung and other cancers. Oncogene 2008, 27(33):4615-4624. 38. Reed SI, Bailly E, Dulic V, Hengst L, Resnitzky D, Slingerland J: G1 control in mammalian cells. J Cell Sci Suppl 1994, 18:69-73. 39. Zhang J, Cicero SA, Wang L, Romito-Digiacomo RR, Yang Y, Herrup K: Nuclear localization of Cdk5 is a key determinant in the postmitotic state of neurons. Proc Natl Acad Sci U S A 2008, 105(25):8772-8777. 40. Zhang J, Li H, Herrup K: Cdk5 nuclear localization is p27-dependent in nerve cells: implications for cell cycle suppression and caspase-3 activation. J Biol Chem 2010, 285(18):14052-14061. 41. Adams JC: Cell-matrix contact structures. Cell Mol Life Sci 2001, 58(3):371-392. 42. Hynes RO: Integrins: bidirectional, allosteric signaling machines. Cell 2002, 110(6):673-687. 43. Mueller SC, Ghersi G, Akiyama SK, Sang QX, Howard L, Pineiro-Sanchez M, Nakahara H, Yeh Y, Chen WT: A novel protease-docking function of integrin at invadopodia. J Biol Chem 1999, 274(35):24947-24952. 44. Sanderson CM, Smith GL: Cell motility and cell morphology: how some viruses take control. Expert Rev Mol Med 1999, 1999:1-16. 45. Orlichenko LS, Radisky DC: Matrix metalloproteinases stimulate epithelial-mesenchymal transition during tumor development. Clin Exp Metastasis 2008, 25(6):593-600. 46. Mazar AP: Urokinase plasminogen activator receptor choreographs multiple ligand interactions: implications for tumor progression and therapy. Clin Cancer Res 2008, 14(18):5649-5655. 47. Rudolph-Owen LA, Chan R, Muller WJ, Matrisian LM: The matrix metalloproteinase matrilysin influences early-stage mammary tumorigenesis. Cancer Res 1998, 58(23):5500-5506. 48. Koblinski JE, Ahram M, Sloane BF: Unraveling the role of proteases in cancer. Clin Chim Acta 2000, 291(2):113-135. 49. Thiery JP: Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer 2002, 2(6):442-454. 50. Humbert S, Dhavan R, Tsai L: p39 activates cdk5 in neurons, and is associated with the actin cytoskeleton. J Cell Sci 2000, 113 ( Pt 6):975-983. 51. Kwon YT, Gupta A, Zhou Y, Nikolic M, Tsai LH: Regulation of N-cadherin-mediated adhesion by the p35-Cdk5 kinase. Curr Biol 2000, 10(7):363-372. 52. Munoz JP, Huichalaf CH, Orellana D, Maccioni RB: cdk5 modulates beta- and delta-catenin/Pin1 interactions in neuronal cells. J Cell Biochem 2007, 100(3):738-749. 53. Kawauchi T, Chihama K, Nabeshima Y, Hoshino M: Cdk5 phosphorylates and stabilizes p27kip1 contributing to actin organization and cortical neuronal migration. Nat Cell Biol 2006, 8(1):17-26. 54. Nakano N, Nakao A, Ishidoh K, Tsuboi R, Kominami E, Okumura K, Ogawa H: CDK5 regulates cell-cell and cell-matrix adhesion in human keratinocytes. Br J Dermatol 2005, 153(1):37-45. 55. Xie Z, Tsai LH: Cdk5 phosphorylation of FAK regulates centrosome-associated miocrotubules and neuronal migration. Cell Cycle 2004, 3(2):108-110. 56. Hou Z, Li Q, He L, Lim HY, Fu X, Cheung NS, Qi DX, Qi RZ: Microtubule association of the neuronal p35 activator of Cdk5. J Biol Chem 2007, 282(26):18666-18670. 57. He L, Hou Z, Qi RZ: Calmodulin binding and Cdk5 phosphorylation of p35 regulate its effect on microtubules. J Biol Chem 2008, 283(19):13252-13260. 58. Zehorai E, Yao Z, Plotnikov A, Seger R: The subcellular localization of MEK and ERK--a novel nuclear translocation signal (NTS) paves a way to the nucleus. Mol Cell Endocrinol 2010, 314(2):213-220. 59. Roberts PJ, Der CJ: Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer. Oncogene 2007, 26(22):3291-3310. 60. Milbrandt J: A nerve growth factor-induced gene encodes a possible transcriptional regulatory factor. Science 1987, 238(4828):797-799. 61. Sukhatme VP, Cao XM, Chang LC, Tsai-Morris CH, Stamenkovich D, Ferreira PC, Cohen DR, Edwards SA, Shows TB, Curran T et al: A zinc finger-encoding gene coregulated with c-fos during growth and differentiation, and after cellular depolarization. Cell 1988, 53(1):37-43. 62. Pignatelli M, Luna-Medina R, Perez-Rendon A, Santos A, Perez-Castillo A: The transcription factor early growth response factor-1 (EGR-1) promotes apoptosis of neuroblastoma cells. Biochem J 2003, 373(Pt 3):739-746. 63. Maegawa M, Arao T, Yokote H, Matsumoto K, Kudo K, Tanaka K, Kaneda H, Fujita Y, Ito F, Nishio K: EGFR mutation up-regulates EGR1 expression through the ERK pathway. Anticancer Res 2009, 29(4):1111-1117. 64. Swirnoff AH, Apel ED, Svaren J, Sevetson BR, Zimonjic DB, Popescu NC, Milbrandt J: Nab1, a corepressor of NGFI-A (Egr-1), contains an active transcriptional repression domain. Mol Cell Biol 1998, 18(1):512-524. 65. Gollob JA, Wilhelm S, Carter C, Kelley SL: Role of Raf kinase in cancer: therapeutic potential of targeting the Raf/MEK/ERK signal transduction pathway. Semin Oncol 2006, 33(4):392-406. 66. McCubrey JA, May WS, Duronio V, Mufson A: Serine/threonine phosphorylation in cytokine signal transduction. Leukemia 2000, 14(1):9-21. 67. Adamson ED, Mercola D: Egr1 transcription factor: multiple roles in prostate tumor cell growth and survival. Tumour Biol 2002, 23(2):93-102.
Cdk5(Cyclin-dependent kinase 5)為CDK家族成員之一,但不具有調控細胞週期運行的功能。此外,Cdk5的專一性活化蛋白並非典型的cyclin而主要是p35蛋白。Cdk5/p35在神經系統與神經退化相關疾病當中扮演重要的角色。在近幾年的研究也發現,Cdk5/p35對於腫瘤細胞的生理調控也相當重要。肺癌一直是全世界發生率以及致死率最高的惡性腫瘤。肺癌依據病理特徵可分為小型細胞肺癌以及非小型細胞肺癌,其中又以非小型細胞肺癌患者佔多數。根據前人研究指出,Cdk5與p35表現量在惡化程度不同的非小型細胞肺癌腫瘤組織中有顯著差異。此外,在韓國族群中發現Cdk5基因促進子(promoter)序列上的單一核苷酸多型性(Single- nucleotide polymorphism,SNP)與肺癌的發生率有正相關的趨勢。但Cdk5和p35參與調控肺癌細胞生長以及生物功能的詳細機制尚未被進一步探討。本篇論文的研究目的在於探討Cdk5和p35於非小型肺癌細胞中所扮演的角色以及調控Cdk5 與p35表現的相關機制。結果顯示,增加Cdk5或是p35的表現可以刺激A549細胞的生長,反之,阻斷Cdk5或是p35的表現則抑制了A549細胞的增生。同時傷口癒合實驗(Wound healing assay)結果顯示抑制了Cdk5或是p35的表現明顯抑制了A549細胞的移動。在神經細胞中,p35的表現被發現受到ERK經由Early Growth Response-1轉錄因子路徑的調控。ERK為表皮生長因子接受器(epidermal growth factor receptor, EGFR)所活化的下游路徑之一。因此我們初步假設在肺癌細胞A549中,EGFR透過活化ERK路徑進而誘導EGR-1以及p35的表現。實驗結果顯示於不同時間點給予A549細胞表皮生長因子(EGF)刺激後,p35的蛋白與messenger RNA表現量隨著時間的上升而增加。利用離體磷酸酶活性分析法 (in vitro kinase assay)進一步檢測也發現EGF能夠促進Cdk5活性上升。為了進一步驗證,我們利用ERK inhibitor(PD98059)以及shEgr-1分別阻斷ERK的活性以及Egr-1的表現,結果顯示EGF誘導p35表現明顯因ERK以及Egr-1受到抑制而阻斷。此外,傷口癒合實驗的結果也顯示,EGF促進A549細胞移動的效應會因Egr-1以及p35的表現受到抑制而阻斷。綜合以上結果,我們發現Cdk5和p35對於非小型肺癌細胞生長與生物功能之調控的重要性,以及在非小型肺癌細胞中p35與Cdk5的表現間接受到EGFR的調控。經由以上研究結果期望能從基礎研究角度充分了解Cdk5和p35對於肺癌的重要性,並對於未來診斷及治療有所貢獻。

Cdk5(Cyclin-dependent kinase 5) belongs to CDK family but does not involve cell cycle regulation. The specific activator of Cdk5 is p35 rather than cyclin. Cdk5/p35 play an important role in nervous system and neurodegenerative disease. In the recent studies, Cdk5/p35 also play an important role in non-neuronal cells including cancer cells. Lung cancer is the leading cause of cancer death in the worldwide. According to the pathological characteristics, lung cancer can be divided into small cell lung cancer and non-small cell lung cancer. Most lung cancer patient are non-small cell lung cancer. According to the previous research, Cdk5/p35 has high expression in non-small cell lung cancer. In addition, single-nucleotide polymorphisms in the promoter of the CDK5 gene contribute to the genetic susceptibility to lung cancer in the Korean population. The mechanism of Cdk5/p35 on cell proliferation and biological function of lung cancer is still unclear. Here we verify the role of Cdk5/p35 in non-small cell lung cancer. The MTT assay results show that Cdk5 or p35 overexpression by transient transfection increased the cell proliferation of A549 cells. On the other hand, knockdown Cdk5 or p35 expression by siRNA could inhibit cell growth and cell migration of A549 cells monitored by wound healing assay. According to the reference published in 2001, ERK induces p35 through induction of Egr-1. ERK is one of the EGFR downstream signal pathways. Here we propose the mechanism of EGFR regulate p35/Cdk5 through ERK/Egr-1 pathway in non-small cell lung cancer. The results show that EGF induces p35 through ERK/Egr-1 pathway in a time-course manner. Cdk5 activity also increased under EGF treatment. Furthermore, the induction of p35 by EGF could be abolished by ERK inhibitor or knockdown Egr-1 by shRNA. The stimulation on cell migration by EGF could be inhibited by knockdown p35 or Egr-1 expression. Taken together, we demonstrate the important of Cdk5/p35 in cell proliferation and cell migration of non-small cell lung cancer. We also demonstrate the regulation of p35/Cdk5 by EGFR through ERK/Egr-1 pathway. Base on these experimental results, we hope to understand the roles of Cdk5/p35 in lung cancer from the view points of basic research. These findings in the future can contribute to the diagnosis and treatment of lung cancer.
其他識別: U0005-2007201114304800
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