肺癌是目前致死率與發生率最高的癌症，尤其是在診斷出病兆時，往往已經是後期或遠端轉移的階段，此時的五年存活率也只剩下約3.9%，因此找到一個可偵測轉移或早期診斷的生物性指標，不管在臨床診斷或抗癌藥物設計中，都是目前重要的議題。
TIMP1是之前研究中找到具有潛力成為偵測轉移的蛋白質之一，因此探討TIMP1的臨床應用與機制為本篇的重點。首先，在機制探討的部分，先比較CL1-0與CL1-5的基本差異。活性表現量可發現五種MMP中有四種MMP有統計上的顯著差異表現量，其中包括MMP2(CL1-5：CL1-0=3.34)和MMP9(CL1-0：CL1-5=2.31)。針對MMP2和MMP9蛋白質與mRNA的表現量差異比較，發現MMP2平均在CL1-5表現量較高(蛋白質是3.35倍, mRNA是6.12倍)，而MMP9平均在CL1-0表現量較高(蛋白質是1.59倍, mRNA是3.13倍)，TIMP1在mRNA的表現量也是在CL1-5表現量高(80.94倍)，三種面向的表現量趨勢一致。由於TIMP1在CL1-5表現量較高，因此欲觀察當降低TIMP1的表現量時，MMP2和MMP9會不會受到影響。所以在這實驗組群中，我們也分為三種面向探討之。在活性表現量的部分，四種MMP中有兩種MMP有統計上的顯著差異表現量，其中只有MMP2(控制組：實驗組=4.21)有差異，而MMP9(控制組：實驗組=1.28)則無。接著，此趨勢分別在蛋白質與mRNA表現量中皆可觀察到，MMP2約相對減少2倍(1.88與1.89)，而MMP9(1.08與0.94)則相對不變。接下來在肺腺癌組織中(N=85) ，發現在遠端轉移(M0/M1), 各期程間(I, II, III, IV)和淋巴分期轉移(N0, N1, N2, N3)都有統計上的差異(P<0.05)。並且在肺腺癌病人(N=188)與健康控制組(N=94)的血液中，觀察到在病人與健康受試者、第一期病人、早期病人(I+II)和晚期病人(III+IV)中，皆看到有表現量的差異。根據以上實驗結果，可推測TIMP1在肺腺癌組織未來臨床應用上，可用來區分轉移與診斷的生物指標，且極有可能是透過調控MMP2的表現量而促進癌細胞的轉移能力。

Lung cancer is the leading cause of cancer death. Most lung cancer patients are diagnosed late; many while having distant metastatic disease; the survival rate drops to around 3.9%. Thus, a biomarker for early diagnosis of metastasis has important clinical implications and is important for anti-cancer drug discovery. The tissue inhibitor of metalloproteinases 1 (TIMP1) is a documented cancer cell invasion-related protein (by previous research). This study will focus on the role of TIMP1 in lung adenocarcinoma metastasis and its clinical implications. In the “mechanism” study, CL1-0 (low invasiveness) and CL1-5 (high invasiveness) cell lines were used as models. In the gelatin zymography test, there were four MMPs with statistically differential activity; this includes MMP2 (CL1-5：CL1-0=3.34) and MMP9 (CL1-0：CL1-5=2.31). MMP2 was of a higher level in CL1-5 (protein level-3.35 fold, mRNA level-6.12 fold). In contrast, MMP9 was of a higher level in CL1-0 (protein level-1.59 fold, mRNA level-3.13 fold). The difference in mRNA levels of TIMP1 for CL1-5 over CL1-0 was 80.94. Because of the higher expression of TIMP1 in CL1-5, we decreased the TIMP1 level in CL1-5 to serve as a model that would allow us to observe the influence on MMP2 and MMP9. Interestingly, the activity of MMP2 was significantly reduced (4.21 fold) but not MMP9 (1.28 fold). The same trend was observed in the protein and mRNA levels of MMP2 (decreased) and MMP9 (unchanged). Tissue samples (N=85) were studied and there was a statistically significant differential expression (p < 0.05) in distant metastasis (I~III and IV), each stage (I, II, III, IV), and lymph node metastasis stages (N0, N1, N2, N3) using tissue microarray (TMA) and immunohistochemistry (IHC). Furthermore, before having enzyme linked immunosorbent assays (ELISA) test, a differential expression of TIMP1 between the plasma of lung adenocarcinoma patients (N=7) and healthy controls (N=2) was observed using Western blot. The data showed that TIMP1 had higher levels in patients than control samples. Based on the results, we tested the plasma samples from 188 patients and 94 healthy controls. TIMP1 was higher in patients, overall stage I (N=97), early stage (I+II, N=129) and late stage (III+IV, N=59). We suggest that TIMP1 is a potential biomarker for the detection of metastasis in the tissue of lung adenocarcinoma patients and promotes cell metastasis through the regulation of MMP2.

Bunn, P. A., Jr., Worldwide overview of the current status of lung cancer diagnosis and treatment. Archives of pathology & laboratory medicine 2012, 136 (12), 1478-81.
Granville, C. A.; Dennis, P. A., An overview of lung cancer genomics and proteomics. American journal of respiratory cell and molecular biology 2005, 32 (3), 169-76.
Hegele, A.; Heidenreich, A.; Kropf, J.; von Knobloch, R.; Varga, Z.; Hofmann, R.; Olbert, P., Plasma levels of cellular fibronectin in patients with localized and metastatic renal cell carcinoma. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine 2004, 25 (3), 111-6.
Schiller, J. H.; Harrington, D.; Belani, C. P.; Langer, C.; Sandler, A.; Krook, J.; Zhu, J.; Johnson, D. H.; Eastern Cooperative Oncology, G., Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer. The New England journal of medicine 2002, 346 (2), 92-8.
Steeg, P. S., Metastasis suppressors alter the signal transduction of cancer cells. Nature reviews. Cancer 2003, 3 (1), 55-63.
Tjalsma, H.; Bolhuis, A.; Jongbloed, J. D.; Bron, S.; van Dijl, J. M., Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome. Microbiology and molecular biology reviews : MMBR 2000, 64 (3), 515-47.
Blanco, M. A.; LeRoy, G.; Khan, Z.; Aleckovic, M.; Zee, B. M.; Garcia, B. A.; Kang, Y., Global secretome analysis identifies novel mediators of bone metastasis. Cell research 2012, 22 (9), 1339-55.
Chiu, K. H.; Chang, Y. H.; Wu, Y. S.; Lee, S. H.; Liao, P. C., Quantitative secretome analysis reveals that COL6A1 is a metastasis-associated protein using stacking gel-aided purification combined with iTRAQ labeling. Journal of proteome research 2011, 10 (3), 1110-25.
Imperlini, E.; Colavita, I.; Caterino, M.; Mirabelli, P.; Pagnozzi, D.; Del Vecchio, L.; Di Noto, R.; Ruoppolo, M.; Orru, S., The secretome signature of colon cancer cell lines. Journal of cellular biochemistry 2013.
Jin, L.; Zhang, Y.; Li, H.; Yao, L.; Fu, D.; Yao, X.; Xu, L. X.; Hu, X.; Hu, G., Differential secretome analysis reveals CST6 as a suppressor of breast cancer bone metastasis. Cell research 2012, 22 (9), 1356-73.
Karagiannis, G. S.; Petraki, C.; Prassas, I.; Saraon, P.; Musrap, N.; Dimitromanolakis, A.; Diamandis, E. P., Proteomic signatures of the desmoplastic invasion front reveal collagen type XII as a marker of myofibroblastic differentiation during colorectal cancer metastasis. Oncotarget 2012, 3 (3), 267-85.
Park, J. E.; Tan, H. S.; Datta, A.; Lai, R. C.; Zhang, H.; Meng, W.; Lim, S. K.; Sze, S. K., Hypoxic tumor cell modulates its microenvironment to enhance angiogenic and metastatic potential by secretion of proteins and exosomes. Molecular & cellular proteomics : MCP 2010, 9 (6), 1085-99.
Wang, C. L.; Wang, C. I.; Liao, P. C.; Chen, C. D.; Liang, Y.; Chuang, W. Y.; Tsai, Y. H.; Chen, H. C.; Chang, Y. S.; Yu, J. S.; Wu, C. C.; Yu, C. J., Discovery of retinoblastoma-associated binding protein 46 as a novel prognostic marker for distant metastasis in nonsmall cell lung cancer by combined analysis of cancer cell secretome and pleural effusion proteome. Journal of proteome research 2009, 8 (10), 4428-40.
Xue, H.; Lu, B.; Lai, M., The cancer secretome: a reservoir of biomarkers. Journal of translational medicine 2008, 6, 52.
Yang, Y.; Lim, S. K.; Choong, L. Y.; Lee, H.; Chen, Y.; Chong, P. K.; Ashktorab, H.; Wang, T. T.; Salto-Tellez, M.; Yeoh, K. G.; Lim, Y. P., Cathepsin S mediates gastric cancer cell migration and invasion via a putative network of metastasis-associated proteins. Journal of proteome research 2010, 9 (9), 4767-78.
Yang, J. C.; Haworth, L.; Sherry, R. M.; Hwu, P.; Schwartzentruber, D. J.; Topalian, S. L.; Steinberg, S. M.; Chen, H. X.; Rosenberg, S. A., A randomized trial of bevacizumab, an anti-vascular endothelial growth factor antibody, for metastatic renal cancer. The New England journal of medicine 2003, 349 (5), 427-34.
Gardner, J.; Ghorpade, A., Tissue inhibitor of metalloproteinase (TIMP)-1: the TIMPed balance of matrix metalloproteinases in the central nervous system. Journal of neuroscience research 2003, 74 (6), 801-6.
Brew, K.; Dinakarpandian, D.; Nagase, H., Tissue inhibitors of metalloproteinases: evolution, structure and function. Biochimica et biophysica acta 2000, 1477 (1-2), 267-83.
Gomez, D. E.; Alonso, D. F.; Yoshiji, H.; Thorgeirsson, U. P., Tissue inhibitors of metalloproteinases: structure, regulation and biological functions. European journal of cell biology 1997, 74 (2), 111-22.
Groft, L. L.; Muzik, H.; Rewcastle, N. B.; Johnston, R. N.; Knauper, V.; Lafleur, M. A.; Forsyth, P. A.; Edwards, D. R., Differential expression and localization of TIMP-1 and TIMP-4 in human gliomas. British journal of cancer 2001, 85 (1), 55-63.
Mayrand, D.; Laforce-Lavoie, A.; Larochelle, S.; Langlois, A.; Genest, H.; Roy, M.; Moulin, V. J., Angiogenic properties of myofibroblasts isolated from normal human skin wounds. Angiogenesis 2012, 15 (2), 199-212.
Nguyen, M.; Arkell, J.; Jackson, C. J., Human endothelial gelatinases and angiogenesis. The international journal of biochemistry & cell biology 2001, 33 (10), 960-70.
Pagenstecher, A.; Stalder, A. K.; Kincaid, C. L.; Shapiro, S. D.; Campbell, I. L., Differential expression of matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase genes in the mouse central nervous system in normal and inflammatory states. The American journal of pathology 1998, 152 (3), 729-41.
Rivera, S.; Tremblay, E.; Timsit, S.; Canals, O.; Ben-Ari, Y.; Khrestchatisky, M., Tissue inhibitor of metalloproteinases-1 (TIMP-1) is differentially induced in neurons and astrocytes after seizures: evidence for developmental, immediate early gene, and lesion response. The Journal of neuroscience : the official journal of the Society for Neuroscience 1997, 17 (11), 4223-35.
Shi, J. H.; Guan, H.; Shi, S.; Cai, W. X.; Bai, X. Z.; Hu, X. L.; Fang, X. B.; Liu, J. Q.; Tao, K.; Zhu, X. X.; Tang, C. W.; Hu, D. H., Protection against TGF-beta1-induced fibrosis effects of IL-10 on dermal fibroblasts and its potential therapeutics for the reduction of skin scarring. Archives of dermatological research 2013, 305 (4), 341-52.
Tan, H. K.; Heywood, D.; Ralph, G. S.; Bienemann, A.; Baker, A. H.; Uney, J. B., Tissue inhibitor of metalloproteinase 1 inhibits excitotoxic cell death in neurons. Molecular and cellular neurosciences 2003, 22 (1), 98-106.
VanMeter, T. E.; Rooprai, H. K.; Kibble, M. M.; Fillmore, H. L.; Broaddus, W. C.; Pilkington, G. J., The role of matrix metalloproteinase genes in glioma invasion: co-dependent and interactive proteolysis. Journal of neuro-oncology 2001, 53 (2), 213-35.
Pesta, M.; Kulda, V.; Kucera, R.; Pesek, M.; Vrzalova, J.; Liska, V.; Pecen, L.; Treska, V.; Safranek, J.; Prazakova, M.; Vycital, O.; Bruha, J.; Holubec, L.; Topolcan, O., Prognostic significance of TIMP-1 in non-small cell lung cancer. Anticancer research 2011, 31 (11), 4031-8.
Peng, Z. M.; Yang, Z. L.; Liu, Y. W., [Expression of MMP1 and TIMP1 proteins in lung cancer and its biological significance]. Hunan yi ke da xue xue bao = Hunan yike daxue xuebao = Bulletin of Hunan Medical University 2002, 27 (2), 159-61.
Aljada, I. S.; Ramnath, N.; Donohue, K.; Harvey, S.; Brooks, J. J.; Wiseman, S. M.; Khoury, T.; Loewen, G.; Slocum, H. K.; Anderson, T. M.; Bepler, G.; Tan, D., Upregulation of the tissue inhibitor of metalloproteinase-1 protein is associated with progression of human non-small-cell lung cancer. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2004, 22 (16), 3218-29.
Fong, K. M.; Kida, Y.; Zimmerman, P. V.; Smith, P. J., TIMP1 and adverse prognosis in non-small cell lung cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 1996, 2 (8), 1369-72.
Simi, L.; Andreani, M.; Davini, F.; Janni, A.; Pazzagli, M.; Serio, M.; Orlando, C., Simultaneous measurement of MMP9 and TIMP1 mRNA in human non small cell lung cancers by multiplex real time RT-PCR. Lung cancer 2004, 45 (2), 171-9.
Jung, Y. S.; Liu, X. W.; Chirco, R.; Warner, R. B.; Fridman, R.; Kim, H. R., TIMP-1 induces an EMT-like phenotypic conversion in MDCK cells independent of its MMP-inhibitory domain. PloS one 2012, 7 (6), e38773.
Ikenaka, Y.; Yoshiji, H.; Kuriyama, S.; Yoshii, J.; Noguchi, R.; Tsujinoue, H.; Yanase, K.; Namisaki, T.; Imazu, H.; Masaki, T.; Fukui, H., Tissue inhibitor of metalloproteinases-1 (TIMP-1) inhibits tumor growth and angiogenesis in the TIMP-1 transgenic mouse model. International journal of cancer. Journal international du cancer 2003, 105 (3), 340-6.
Iniesta, P.; Moran, A.; De Juan, C.; Gomez, A.; Hernando, F.; Garcia-Aranda, C.; Frias, C.; Diaz-Lopez, A.; Rodriguez-Jimenez, F. J.; Balibrea, J. L.; Benito, M., Biological and clinical significance of MMP-2, MMP-9, TIMP-1 and TIMP-2 in non-small cell lung cancer. Oncology reports 2007, 17 (1), 217-23.
Lim, B. J.; Jung, S. S.; Choi, S. Y.; Lee, C. S., Expression of metastasis-associated molecules in non-small cell lung cancer and their prognostic significance. Molecular medicine reports 2010, 3 (1), 43-9.
Xiao, T.; Ying, W.; Li, L.; Hu, Z.; Ma, Y.; Jiao, L.; Ma, J.; Cai, Y.; Lin, D.; Guo, S.; Han, N.; Di, X.; Li, M.; Zhang, D.; Su, K.; Yuan, J.; Zheng, H.; Gao, M.; He, J.; Shi, S.; Li, W.; Xu, N.; Zhang, H.; Liu, Y.; Zhang, K.; Gao, Y.; Qian, X.; Cheng, S., An approach to studying lung cancer-related proteins in human blood. Molecular & cellular proteomics : MCP 2005, 4 (10), 1480-6.
Hoikkala, S.; Paakko, P.; Soini, Y.; Makitaro, R.; Kinnula, V.; Turpeenniemi-Hujanen, T., Tissue MMP-2 and MMP-9 [corrected] are better prognostic factors than serum MMP-2/TIMP-2--complex or TIMP-1 [corrected] in stage [corrected] I-III lung carcinoma. Cancer letters 2006, 236 (1), 125-32.
Page-McCaw, A.; Ewald, A. J.; Werb, Z., Matrix metalloproteinases and the regulation of tissue remodelling. Nature reviews. Molecular cell biology 2007, 8 (3), 221-33.
Gross, J. L., C. M., Collagenolytic activity in amphibian tissues: a tissue culture assay. Proc Natl Acad Sci U S A. 1962, 48, 1014-22.
Cerezo, M.; Tichet, M.; Abbe, P.; Ohanna, M.; Lehraiki, A.; Rouaud, F.; Allegra, M.; Giacchero, D.; Bahadoran, P.; Bertolotto, C.; Tartare-Deckert, S.; Ballotti, R.; Rocchi, S., Metformin blocks melanoma invasion and metastasis development in a AMPK/p53-dependent manner. Molecular cancer therapeutics 2013.
Hong, J. S.; Greenlee, K. J.; Pitchumani, R.; Lee, S. H.; Song, L. Z.; Shan, M.; Chang, S. H.; Park, P. W.; Dong, C.; Werb, Z.; Bidani, A.; Corry, D. B.; Kheradmand, F., Dual protective mechanisms of matrix metalloproteinases 2 and 9 in immune defense against Streptococcus pneumoniae. Journal of immunology 2011, 186 (11), 6427-36.
Kim, Y. M.; Kim, I. H.; Nam, T. J., Inhibition of AGS human gastric cancer cell invasion and proliferation by Capsosiphon fulvescens glycoprotein. Molecular medicine reports 2013, 8 (1), 11-6.
Kou, Y.; Inaba, H.; Kato, T.; Tagashira, M.; Honma, D.; Kanda, T.; Ohtake, Y.; Amano, A., Inflammatory responses of gingival epithelial cells stimulated with Porphyromonas gingivalis vesicles are inhibited by hop-associated polyphenols. Journal of periodontology 2008, 79 (1), 174-80.
Liu, Q.; Sun, Y.; Rihn, S.; Nolting, A.; Tsoukas, P. N.; Jost, S.; Cohen, K.; Walker, B.; Alter, G., Matrix metalloprotease inhibitors restore impaired NK cell-mediated antibody-dependent cellular cytotoxicity in human immunodeficiency virus type 1 infection. Journal of virology 2009, 83 (17), 8705-12.
Shimizu-Hirota, R.; Xiong, W.; Baxter, B. T.; Kunkel, S. L.; Maillard, I.; Chen, X. W.; Sabeh, F.; Liu, R.; Li, X. Y.; Weiss, S. J., MT1-MMP regulates the PI3Kdelta.Mi-2/NuRD-dependent control of macrophage immune function. Genes & development 2012, 26 (4), 395-413.
Yen, C. Y.; Liang, S. S.; Han, L. Y.; Chou, H. L.; Chou, C. K.; Lin, S. R.; Chiu, C. C., Cardiotoxin III Inhibits Proliferation and Migration of Oral Cancer Cells through MAPK and MMP Signaling. TheScientificWorldJournal 2013, 2013, 650946.
Zhang, J.; Zhang, D.; Wu, G. Q.; Feng, Z. Y.; Zhu, S. M., Propofol inhibits the adhesion of hepatocellular carcinoma cells by upregulating microRNA-199a and downregulating MMP-9 expression. Hepatobiliary & pancreatic diseases international : HBPD INT 2013, 12 (3), 305-9.
Newby, A. C., Dual role of matrix metalloproteinases (matrixins) in intimal thickening and atherosclerotic plaque rupture. Physiol Rev. 2005, 85 (1), 1-31.
Shah, P. K., Inflammation, metalloproteinases, and increased proteolysis: an emerging pathophysiological paradigm in aortic aneurysm. Circulation 1997, 96 (7), 2115-7.
Spinale, F. G., Matrix Metalloproteinases: Regulation and Dysregulation in the Failing Heart. Circulation research 2002, 90 (5), 520-530.
Visse, R.; Nagase, H., Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circulation research 2003, 92 (8), 827-39.
Allen, D. L.; Teitelbaum, D. H.; Kurachi, K., Growth factor stimulation of matrix metalloproteinase expression and myoblast migration and invasion in vitro. American journal of physiology. Cell physiology 2003, 284 (4), C805-15.
Nakamura, T. S., A.; Ishikawa, T., Effects of hyaluronan on matrix metalloproteinase activity in skin fibroblasts. International Congress Series 2001, 1223, 259–263.
Pirilä, E.; Sharabi, A.; Salo, T.; Quaranta, V.; Tu, H.; Heljasvaara, R.; Koshikawa, N.; Sorsa, T.; Maisi, P., Matrix metalloproteinases process the laminin-5 2-chain and regulate epithelial cell migration. Biochemical and Biophysical Research Communications 2003, 303 (4), 1012-1017.
Ronziere, M. C.; Aubert-Foucher, E.; Gouttenoire, J.; Bernaud, J.; Herbage, D.; Mallein-Gerin, F., Integrin alpha1beta1 mediates collagen induction of MMP-13 expression in MC615 chondrocytes. Biochimica et biophysica acta 2005, 1746 (1), 55-64.
Ulku, A. S.; Schafer, R.; Der, C. J., Essential role of Raf in Ras transformation and deregulation of matrix metalloproteinase expression in ovarian epithelial cells. Molecular cancer research : MCR 2003, 1 (14), 1077-88.
Shimokawa Ki, K.; Katayama, M.; Matsuda, Y.; Takahashi, H.; Hara, I.; Sato, H.; Kaneko, S., Matrix metalloproteinase (MMP)-2 and MMP-9 activities in human seminal plasma. Molecular human reproduction 2002, 8 (1), 32-6.