肺癌為全世界癌症死因中排名順位第一位，且診斷出來的時候往往已進入癌症晚期且預後不佳，其五年存活率僅16%，而癌細胞的轉移更是造成肺癌難以治療的主因。酪胺酸激酶在肺癌中被發現與癌症的轉移跟入侵有關，並根據先前的研究，在CL1-5中找到的酪胺酸磷酸化蛋白質經由Motif分析後，發現與Src激酶、EGFR激酶及JAK激酶相關，本篇研究挑選了Dasatinib、Gefitinib、JAK inhibitor1三個酪胺酸抑制劑，經由細胞功能性試驗，篩選出Dasatinib作為本實驗所添加的抑制劑，接著使用非標記定量分析策略去找尋受到酪胺酸抑制劑抑制後與轉移有關的酪胺酸磷酸化蛋白質，因此去比較CL1-5細胞與CL1-5細胞受到酪胺酸抑制劑Dasatinib抑制後，兩組別中所鑑定到與轉移相關的酪胺酸磷酸化蛋白質。首先將CL1-5細胞與CL1-5細胞處理Dasatinib的樣本進行免疫沉澱法，藉此將樣本中酪胺酸磷酸化蛋白質純化下來，接著利用stacking gel膠體電泳，除去干擾物後並以本實驗室發展出的分析策略，並配合iPhos軟體，在圖譜中找尋到相差79.966Da訊號。找到419個可能的磷酸化訊號並利用targeted MS/MS分析鑑定到491個酪胺酸磷酸化蛋白質，接著使用非標記定量分析，有472(96%)個酪胺酸磷酸化蛋白質被定量，並由統計分析的結果，取p<0.01為在CL1-5細胞與CL1-5細胞處理Dasatinib兩組別中有差異的酪胺酸磷酸化蛋白質，其中有67個在CL1-5細胞處理Dasatinib中有高表現量，而有36個在CL1-5細胞中有高表現量，最後將這103差異表現的酪胺酸磷酸化蛋白質利用Motif-X軟體分析並比對可能的激酶，有五個發現與肺癌轉移相關，而利用MetaCore來進行酪胺酸蛋白質的交互作用分析比對，發現有40個酪胺酸磷酸蛋白質皆與c-Myc相關，而c-Myc為一個非小細胞肺癌有關的轉移基因，由此資訊可以提供未來進一步去了解肺癌轉移的新方向。

Lung cancer is a lethal disease and early metastasis is the major cause of treatement failure and cancer-related death. Tyrosine kinase is involved in the invasive and metastic behaviors of lung cancer. According to current research result, showed tyrosine phosphorylation altered levels between CL1-0 and CL1-5 and extracted motifs for Src、EGFR、JAK2 kinases. First we use cell functional assay to test Dasatinib、Gefitinib and JAK inhibitor1 three inhibitors and final select Dasatinib to treate CL1-5 cells. The aim of this research is to identify metastasis related phosphotyrosine proteins in response to tyrosine kinase inhibitor treatment by label-free quantitative analysis.Tyrosine phophorylated proteins were immunoprecipitated from CL1-5 and CL1-5 with Dasatinib treatment and resolved on a stacking gel. The comprehensive characterization of both CL1-5 and CL1-5 with Dasatinib treatment tyrosine phosphoproteome was achieved by our developed strategy and combined with iPhos program. We have identified 419 peak signals and 491 tyrosine phosphoproteins are identified by targeted MS/MS. 473(96%) are quantified by label-free quantitative analysis. Following, the statistical result and cut-off p-value≦0.01 as differentially expressed, 67 tyrosine phosphoproteins have higher level in CL1-5 cells with Dasatinib treatment while 36 with higher levels in CL1-5 cells. Those 103 differentially expressed tyrosine phosphoproteins converge toward six motifs and five kinases or phosphatase substracts are related to lung cancer metastasis. Moreover after interactome analysis by MetaCore software which point to c-Myc that is a metastasis gene for non-small-cell lung cancer and this information may provide us a direction to understand lung cancer metastasis.

Alvarez JV, Greulich H, Sellers WR, et al. 2006. Signal transducer and activator of transcription 3 is required for the oncogenic effects of non-small-cell lung mutations of the epidermal growth factor receptor. Cancer Res 66:3162-3168.

Asante AE, Ball K, Bateman K, et al. 2001. The YRD motif is a major determinant of substrate and inhibitor specificity in T-cell protein-tyrosine phosphatase. J. Biol. Chem. 276:26036-26043.

Ballif B A, Carey GR, Sunyaev SR, et al. 2008. Largescale identification and evolution indexing of tyrosine phosphorylation sites from murine brain. J. Proteome Res 7:311-318 311-318.

Baker S J, Rane SG, Reddy EP, et al. 2007. Hematopoietic cytokine receptor signaling. Oncogene 26:6724-6737.

Bantscheff M, Schirle M, Sweetman G, et al. 2007. Quantitative mass spectrometry in proteomics: a critical review. Anal Bioanal Chem 389:1017-1031.

Bergers G, Benjamin LE. 2003. Tumorigenesis and the angiogenic switch. Nature reviews 3:401-410.

Bergström LS, Molin M, Savitski MM, et al. 2008. Immunoaffinity enrichments followed by mass spectrometric detection for studying global protein tyrosine phosphorylation. J. Proteome. Res. 7:2897-2910.

Blume JP, Hunter T. 2001. Oncogenic kinase signalling. Nature 411:355-365.

Bromann PA, Korkaya H, Courtneidge SA. 2004. The interplay between Src family kinases and receptor tyrosine kinases. Oncogene. 23:7957-7968.

Burgess MR, Skaggs BJ, Shah NP, et al. 2005. Comparative analysis of two clinically active BCRABL kinase inhibitors reveals the role of conformationspecific binding in resistance. Proc Natl Acad Sci USA 102:3395-3400.
Burridge K, Sastry SK, Sallee JL. 2006. Regulation of cell adhesion by protein-tyrosine phosphatases.1. Cellmatrix adhesion. J Biol Chem 281:15593-15596.

Calvo A, Xiao N, Kang J, et al. 2002. Alterations in gene expression profiles during prostate cancer progression: functional correlations to tumorigenicity and downregulation of selenoprotein-P in mouse and human tumors. Cancer Res 62: 5325-5335.

Chen JJ, Peck K, Hong TM, et al. 2001. Global analysis of gene expression in invasion by a lung cancer model. Cancer research 61:5223-5230.

Chu YW, Yang PC, Yang SC, et al. 1997. Selection of invasive and metastatic subpopulations from a human lung adenocarcinoma cell line. Am. J. Respir. Cell Mol. Biol. 17:353-360.

Clevenger CV. 2004. Roles and regulation of stat family transcription factors in human breast cancer. Am.J. Pathol 165:1449-1460.

De PF, Granato AM, Scarpi E, et al. 2002. Vascular endothelial growth factor and prognosis in patients with node-negative breast cancer. Int J Cancer 98:228-233.

Fontanini G, Vignati S, Bigini D, et al. 1995. Epidermal growth factor receptor (EGFr) expression in non–small cell lung carcinomas correlates with metastatic involvement of hilar and mediastinal lymph nodes in the squamous subtype. Eur. J. Cancer 31A:178-183.

Frame MC. 2002. Src in cancer: deregulation and consequences for cell behaviour. Biochim Biophys Acta 1602:114–130.

Fry WA, Phillips JL, Menck HR. 1999. Ten-year survey of lung cancer treatment and survival in hospitals in the United States: a national cancer data base report. Cancer 86:1867-1876.

Gridelli C, Langer C, Maione P, et al. 2007. Lung cancer in the elderly. J Clin Oncol 25:1898-1907.

Haqqani AS, Kelly JF, Stanimirovic DB, et al. 2008. Quantitative protein profiling by mass spectrometry using label-free proteomics. Methods Mol Biol 439:241-256.

Haura EB, Zheng Z, Song L, et al. 2005. Cantor A and Bepler G: Activated epidermal growth factor receptor-Stat-3 signaling promotes tumor survival in vivo in non-small cell lung cancer. Clin Cancer Res 11:8288-8894.

Hunter T. 2000. Signaling - 2000 and beyond. Cell 100:113-127.

Hunter T, Sudarsanam S. 2002. The protein kinase complement of the human genome. Science. 298:1912-1934.

Irby RB, Yeatman TJ. 2000. Role of Src expression and activation in human cancer.
Oncogene 19:5636-5642.

Johnson FM, Saigal B, Talpaz M, et al.2005. Dasatinib (BMS-354825) tyrosine kinase inhibitor suppresses invasion and induces cell cycle arrest and apoptosis of head and neck squamous cell carcinoma and non-small cell lung cancer cells. Clin Cancer Res 11:6924-6932.

Kim YN, Dam P, Bertics PJ, et al. 2002. Caveolin-1 phosphorylation in human squamous and epidermoid carcinoma cells: dependence on ErbB1 expression and Src activation. Exp. Cell Res 280:134-147.

Larsen M, Tremblay ML, Yamada KM. 2003. Phosphatases in cell-matrix adhesion and migration. Nat Rev Mol Cell Biol 4:700-711.

Lim YP. 2005. Mining the tumor phosphoproteome for cancer markers. Clin Cancer Res 11:3163-3169.

Lombardo LJ, Lee FY, Chen P, et al. 2004. Discovery of N-(2-chloro-6-methyl-phenyl)-2-(6-(4-(2-hydroxyethyl)-piperazin-1-yl)-2-methylpyrimidin-4-ylamino)thiazole-5-carboxamide (BMS-354825), a dual Src/Abl kinase inhibitor with potent antitumor activity in preclinical assays. JMed Chem 47:6658-6661.

Masaki T, Igarashi K, Tokuda M, et al. 2003. pp60c-src activation in lung adenocarcinoma. Eur. J. Cancer 39:1447-1455.
Nam S, Kim D, Cheng JQ, et al. 2005. Action of the Src familykin ase inhibitor, dasatinib (BMS-354825), on human prostate cancer cells. Cancer Res. 65: 9185-9189.

Ostman A, Hellberg C, Bohmer FD. 2006. Protein-tyrosine phosphatases and cancer. Nat Rev Cancer 6:307-320.

Pandey A, Fernandez MM, SteenH, et al. 2000. Identification of a novel immunoreceptor tyrosine-based activation motif-containing molecule, STAM2, by mass spectrometry and its involvement in growth factor and cytokine receptor signaling pathways. J. Biol. Chem 275:38633-38639.

Pandey A, Podtelejnikov AV, Blagoev B, et al. 2000. Analysis of receptor signaling pathways by mass spectrometry: identification of vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors. Proc. Natl. Acad. Sci. USA 97:179-184.

Parkin DM. 2001. Global cancer statistics in the year 2000. Lancet Oncol 2:533-543.

Jemal A, Murray T, Samuels A, et al. 2003. Cancer statistics. CA Cancer J Clin 53:5-26.

Parsons SJ, Parsons JT. 2004. Src family kinases, key regulators of signal transduction.
Oncogene 23:7906-7909.

Reinders J, Sickmann A. 2005. State-of-the-art in phosphoproteomics. Proteomics 5:4052-4061.

Robinson DR, Wu YM, Lin SF. 2000. The protein tyrosine kinase family of the human genome. Oncogene 19:5548-5557.

Roskoski JR. 2004. Src protein-tyrosine kinase structure and regulation. Biochem
Biophys Res Commun 324:1155-1164.

Roskoski JR. 2005. Src kinase regulation by phosphorylation and dephosphorylation.Biochem Biophys Res Commun 331:1-14.

Salido M, Pijuan L, Martinez AL. 2011. Increased ALK gene copy number and amplification are frequent in non-small cell lung cancer. J Thorac Oncol 6:21-27.
Salomon AR, Ficarro SB, Brill LM, et al. 2003. Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry. Proc. Natl. Acad. Sci. USA 100: 443-448.

Schwartz D, Gygi SP. 2005. An iterative statistical approach to the identification of protein phosphorylation motifs from large-scale data sets. Nat. Biotechnol 23:1391-1398.

Shah NP, Tran C, Lee FY, et al. 2004. Overriding imatinib resistance with a novel ABL kinase inhibitor. Science 305:399-401.

Shor AC, Keschman EA, Lee FY, et al. 2007. Dasatinib inhibits migration and invasion in diverse human sarcoma cell lines and induces apoptosis in bone sarcoma cells dependent on Src kinase for survival. Cancer Res 67:2800-2808.

Sickmann A, Mreyen M, Meyer HE, et al. 2002. Identification of modified proteins by mass spectrometry. IUBMB Life 54:51-57.

Schlessinger J. 2000. New roles for Src kinases in control of cell survival and angiogenesis. Cell 100:293-296.

Silver DL, Montell DJ. 2001. Paracrine signaling through the JAK/STAT pathway activates invasive behavior of ovarian epithelial cells in Drosophila. Cell 107:831-841.

Song L, Morris M, Bagui T, et al. 2006. Dasatinib (BMS-354825) selectively induces apoptosis in lung cancer cells dependent on epidermal growth factor receptor signaling for survival. Cancer Res 66:5542-5548.
Songyang Z, Cantley LC. 1995. Recognition and specificity in protein tyrosine kinase-mediated signaling. Trends Biochem. Sci 20:470-475.

Sugiyama N, Masuda T, Shinoda K, et al. 2007. Phosphopeptide enrichment by aliphatic hydroxyl acid-modified metal oxide chromatography for nano-LCMS/MS in proteomics applications. Mol. Cell. Proteomics 6:1103-1109.

Summy JM, Gallick GE. 2003. Src family kinases in tumor progression and metastasis. Cancer Metastasis Rev 22:337-358.

Su HT, Chen YM, Perng RP. 2008. Symptomatic ocular metastases in lung cancer. Respirology Carlton, Vic 13:303-305.

Ulf RR, Christian K, Fatih C, et al. 2009. Myc is a metastasis gene for non-small-cell lung cancer. PLoS ONE 4:e6029

Rush J, Moritz A, Lee KA, et al. 2005. Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nat. Biotechnol 23:94-101.

Volm M, Drings P, Wodrich W, et al. 1993. Expression of oncoprotein in primary human non-small cell lung cancer and incidence of metastasis. Clin Exp Metastasis 11: 325-329.

Wong JW, Sullivan MJ, Cagney G, et al. 2008. Computational methods for the comparative quantification of proteins in label-free LCn-MS experiments. Brief Bioinform 9:156-165.

Wu HY, Tseng VS, Chen LC, et al. 2010. Identification of tyrosine-phosphorylated proteins associated with lung cancer metastasis using label-free quantitative analyses. J Proteome Res 9:4102-4112.

Wu HY., Tseng V SM, Chen LC., et al. 2009. Combining Alkaline Phosphatase Treatment and LTQ/Orbitrap High Mass Accuracy LC-MS Data for the Efficient and Confident Identification of Protein Phosphorylation. Analytical Chemistry 81:7778-7787.

Wu HY, Huang FY, Chang YC, et al. 2008 Strategy for determination of in vitro protein acetylation sites by using isotope-labeled acetyl coenzyme A and liquid chromatography-mass spectrometry. Analytical Chemistry 80:6178-6189.

Wu WW, Wang G, Baek SJ, et al. 2006. Comparative study of three proteomic quantitative methods, DIGE, cICAT, and iTRAQ, using 2D gel- or LC-MALDI TOF/TOF. J Proteome Res 5:651-658.

Yeatman TJ. 2004. A renaissance for SRC. Nat Rev Cancer 4:470-480.

Yu H, Jove R. 2004. The STATs of cancer - new molecular targets come of age. Nat Rev 4:97-105.
Zahedi RP, Begonja AJ, Gambaryan S, et al. 2006. Phosphoproteomics of human platelets: a quest for novel activation pathways. Biochim. Biophys. Acta 1764:1963-1976.

Zhang J, Kalyankrishna S, Wislez M, et al. 2007. SRC-family kinases are activated
in non-small cell lung cancer and promote the survival of epidermal growth factor receptor-dependent cell lines. Am J Pathol 170:366-376.

Zhang Q, Thomas SM, Xi S, et al.2004. SRC family kinases mediate epidermal growth factor receptor ligand cleavage, proliferation, and invasion of head and neck cancer cells. Cancer Res 64:6166-61731.

Zhao M, Gao FH, Wang JY, et al. 2011. JAK2/STAT3 signaling pathway activation mediates tumor angiogenesis by upregulation of VEGF and bFGF in non-small-cell lung cancer. Lung cancer 73:366-374.

Zheng R, Yano S, Matsumori Y, et al. 2005. Src tyrosine kinase inhibitor, M475271, suppresses subcutaneous growth and production of lung metastasis via inhibition of proliferation, invasion, and vascularization of human lung adenocarcinoma cells. Clin Exp Metastasis V22:195-204.