EGF receptor tyrosine kinase substrate no.8 (Eps8)是一個致癌蛋白，參與人類癌症的發展與進程。而Insulin receptor tyrosine kinase substrate protein of 53kDa (IRSp53)是Eps8的結合蛋白，他們之間的交互作用是透過IRSp53的SH3 domain和WWB domain與Eps8的SH3 domain和proline-rich region做結合。目前已有六個IRSp53的異購物被發現，在此我們命為IRSp53-S，IRSp53-L，IRSp53-T，IRSp58-M，IRSp53-SCt和IRSp53-S+A。至今IRSp53異構物對於大腸癌的發展調控作用仍是未知，因此我們想要進一步去探討IRSp53的異構物對於大腸癌細胞有何重要性。從實驗結果中我們發現IRSp53-S的mRNA在HT29，SW480以及SW620大腸癌細胞中的表現量最一致並占了很大一部分，在大腸癌病患的檢體中同樣也發現IRSp53-S的高表現結果。但IRSp53-S的mRNA與蛋白質在細胞中的表現卻不一致。在SW480細胞內，IRSp53-S的mRNA與SW620 細胞是相當的，但是其IRSp53-S 蛋白是非常的少。由於IRSp53-S的蛋白穩定度在SW480細胞並不比在SW620細胞低，我們推測可能是跟後轉錄修飾的蛋白生成調控有關。在生物學功能的研究上，我們發現IRSp53-S和IRSp58-M會增加SW480大腸癌細胞的移動能力；相反的，IRSp53-T和IRSp53-SCt會抑制SW480大腸癌細胞的移動能力。從以上結果，我們認為，不同的IRSp53異構物對於大腸癌細胞的進展扮演著不同的調控功能。

EGF receptor tyrosine kinase substrate no.8 (Eps8) is an oncoprotein. Its level has been linked to human cancer development and progression. Insulin receptor tyrosine kinase substrate protein of 53kDa (IRSp53) is one of the Eps8 binding proteins. The interaction between IRSp53 and Eps8 is mediated by the C-terminal SH3/WWB domain in IRSp53 and the SH3/proline-rich region in Eps8. Currently, six isoforms (IRSp53-S, IRSp53-L, IRSp53-T, IRSp58-M, IRSp53-SCt and IRSp53-S+A) have been identified in human cells. However, the role of these isoforms in cancer progression remains unknown. In this study, through qPCR analysis, we found that the mRNA level of IRSp53-S is the most abundant form of IRSp53 in HT29, SW480 and SW620 cell lines as well as in clinical colorectal carcinoma. Surprisingly, the protein expression of IRSp53-S is not correlated with its mRNA level in the tested colon cancer cells, such that the protein level of IRSp53-S is dramtically decreased in SW480 cells while its mRNA level is similar in between SW480 and SW620 cells. This down-regulation of IRSp53-S is not due to the decreased protein stability in SW480 cells since the turn-over rate of IRSp53-S is faster in SW620 cells than in SW480 cells. In cell motility study, our data indicate that IRSp53-S and IRSp58-M play a positive role while IRSp53-T and IRSp53-SCt play a negative role in regulation cell movement in SW480 cells. Our results highlight that each IRSp53 isoform may play differential roles in regulating colon cancer cell progression.

1 Fearnhead NS, Britton MP, Bodmer WF. The ABC of APC. Human Molecular Genetics 2001; 10: 721-733.

2 Arnold CN, Goel A, Blum HE, Boland CR. Molecular pathogenesis of colorectal cancer: implications for molecular diagnosis. Cancer 2005; 104: 2035-2047.

3 Jasperson KW, Tuohy TM, Neklason DW, Burt RW. Hereditary and familial colon cancer. Gastroenterology 2010; 138: 2044-2058.

4 Amersi F, Agustin M, Ko CY. Colorectal Cancer: Epidemiology, Risk Factors, and Health Services. Clinics In Colon And Rectal Surgery 2005; 18: 133-140.

5 Fearon EF, Vogelstein B. A Genetic Model for Colorectal Tumorigenesis. Cell 1990; 61: 759-767.

6 Rivlin N, Brosh R, Oren M, Rotter V. Mutations in the p53 Tumor Suppressor Gene: Important Milestones at the Various Steps of Tumorigenesis. Genes Cancer 2011; 2: 466-474.

7 Ramos M, Franch P, Zaforteza M, Artero J, Duran M. Completeness of T, N, M and stage grouping for all cancers in the Mallorca Cancer Registry. BMC Cancer 2015; 15: 847.

8 Edge SB, Compton CC. The American Joint Committee on Cancer: the 7th edition of the AJCC cancer staging manual and the future of TNM. Ann Surg Oncol 2010; 17: 1471-1474.

9 Goldberg RM, Sargent DJ, Morton RF, Fuchs CS, Ramanathan RK, Williamson SK et al. A randomized controlled trial of fluorouracil plus leucovorin, irinotecan, and oxaliplatin combinations in patients with previously untreated metastatic colorectal cancer. J Clin Oncol 2004; 22: 23-30.

10 Tournigand C, Andre T, Achille E, Lledo G, Flesh M, Mery-Mignard D et al. FOLFIRI followed by FOLFOX6 or the reverse sequence in advanced colorectal cancer: a randomized GERCOR study. J Clin Oncol 2004; 22: 229-237.

11 Heinemann V, Douillard JY, Ducreux M, Peeters M. Targeted therapy in metastatic colorectal cancer -- an example of personalised medicine in action. Cancer Treat Rev 2013; 39: 592-601.

12 Zouhairi ME, Charabaty A, Pishvaian MJ. Molecularly Targeted Therapy for Metastatic Colon Cancer: Proven Treatments and Promising New Agents. Gastrointestinal Cancer Research 2011; 4 15-21.

13 Mayer RJ, Van Cutsem E, Falcone A, Yoshino T, Garcia-Carbonero R, Mizunuma N et al. Randomized trial of TAS-102 for refractory metastatic colorectal cancer. N Engl J Med 2015; 372: 1909-1919.

14 Yeh TC, Ogawa W, Danielsen AG, Roth RA. Characterization and Cloning of a 58/53-kDa Substrate of the Insulin Receptor Tyrosine Kinase*. The Journal of biological chemistry 1996; 271: 2921-2928.

15 Oda K, Shiratsuchi T, Nishimori H, Inazawa J, Yoshikawa H, Taketani Y et al. Identification of BAIAP2 (BAI-associated protein 2), a novel human homologue of hamster IRSp53, whose SH3 domain interacts with the cytoplasmic domain of BAI1. Cytogenet Cell Genet 1999; 84: 75-82.

16 Miyahara A, Okamura-Oho Y, Miyashita T, Hoshika A, Yamada M. Genomic structure and alternative splicing of the insulin receptor tyrosine kinase substrate of 53-kDa protein. J Hum Genet 2003; 48: 410-414.

17 Mattila PK, Pykalainen A, Saarikangas J, Paavilainen VO, Vihinen H, Jokitalo E et al. Missing-in-metastasis and IRSp53 deform PI(4,5)P2-rich membranes by an inverse BAR domain-like mechanism. J Cell Biol 2007; 176: 953-964.

18 Yamagishi A, Masuda M, Ohki T, Onishi H, Mochizuki N. A novel actin bundling/filopodium-forming domain conserved in insulin receptor tyrosine kinase substrate p53 and missing in metastasis protein. The Journal of biological chemistry 2004; 279: 14929-14936.

19 Millard TH, Bompard G, Heung MY, Dafforn TR, Scott DJ, Machesky LM et al. Structural basis of filopodia formation induced by the IRSp53/MIM homology domain of human IRSp53. The EMBO Journal 2005; 24: 240-250.

20 Govind S, Kozma R, Monfries C, Lim L, Ahmed S. Cdc42Hs Facilitates Cytoskeletal Reorganization and Neurite Outgrowth by Localizing the 58-kD Insulin Receptor Substrate to Filamentous Actin. The Journal of Cell Biology 2001; 152: 579-594.

21 Bockmann J, Kreutz MR, Gundelfinger ED, Bo¨ckers TM. ProSAP/Shank postsynaptic density proteins interact with insulin receptor tyrosine kinase substrate IRSp53. Journal of Neurochemistry 2002; 83: 1013-1017.

22 Sekerkova G, Loomis PA, Changyaleket B, Zheng L, Eytan R, Chen B et al. Novel Espin Actin-Bundling Proteins Are Localized to Purkinje Cell Dendritic Spines and Bind the Src Homology 3 Adapter Protein Insulin Receptor Substrate p53. The Journal of Neuroscience 2003; 23: 1310-1319.

23 Fujiwara T, Mammoto A, Kim Y, Takai Y. Rho small G-protein-dependent binding of mDia to an Src homology 3 domain-containing IRSp53/BAIAP2. Biochem Biophys Res Commun 2000; 271: 626-629.

24 Funato Y, Terabayashi T, Suenaga N, Seiki M, Takenawa T, Miki H. IRSp53/Eps8 Complex Is Important for Positive Regulation of Rac and Cancer Cell Motility/Invasiveness. CANCER RESEARCH 2004; 64: 5237-5244.

25 Liu PS, Jong TH, Maa MC, Leu TH. The interplay between Eps8 and IRSp53 contributes to Src-mediated transformation. Oncogene 2010; 29: 3977-3989.

26 Kast DJ, Yang C, Disanza A, Boczkowska M, Madasu Y, Scita G et al. Mechanism of IRSp53 inhibition and combinatorial activation by Cdc42 and downstream effectors. Nature structural & molecular biology 2014; 21: 413-422.

27 Disanza A, Mantoani S, Hertzog M, Gerboth S, Frittoli E, Steffen A et al. Regulation of cell shape by Cdc42 is mediated by the synergic actin-bundling activity of the Eps8-IRSp53 complex. Nat Cell Biol 2006; 8: 1337-1347.

28 Fazioli F, Minichiello L, Matoska V, Castagnino P, Miki T, T.Wong W et al. Eps8, a substrate for the epidermal growth factor receptor kinase, enhances EGF-dependent mitogenic signals. The EMBO Journal 1993; 12: 3799-3808.

29 Maa M-C, Lai J-R, Lin R-W, Leu T-H. Enhancement of tyrosyl phosphorylation and protein expression of eps8 by v-Src. Biochimica et Biophysica Acta 1999; 1450: 341-351.

30 Maa M-C, Hsieh C-Y, Leu T-H. Overexpression of p97Eps8 leads to cellular transformation: implication of pleckstrin homology domain in p97Eps8-mediated ERK activation. Oncogene 2001; 20: 106-112.

31 Fiore PPD, Scita G. Eps8 in the midst of GTPases. The International Journal of Biochemistry & Cell Biology 2002; 34: 1178-1183.

32 Biesova Z, Piccoli C, Wong WT. Isolation and characterization of e3B1, an eps8 binding protein that regulates cell growth. Oncogene 1997; 14: 233-241.

33 Scita G, Nordstrom J, Carbone R, Tenca P, Giardina G, Gutkind S et al. EPS8 and E3B1 transduce signals from Ras to Rac. Nature 1999; 401: 290-293.

34 Disanza A, Carlier MF, Stradal TE, Didry D, Frittoli E, Confalonieri S et al. Eps8 controls actin-based motility by capping the barbed ends of actin filaments. Nat Cell Biol 2004; 6: 1180-1188.

35 Wang W, Wyckoff JB, Frohlich VC, Oleynikov Y, ttelmaier SH, Zavadil J et al. Single Cell Behavior in Metastatic Primary Mammary Tumors Correlated with Gene Expression Patterns Revealed by Molecular Profiling. Cancer Research 2002; 62: 6278-6288.

36 Chen YJ, Shen MR, Chen YJ, Maa MC, Leu TH. Eps8 decreases chemosensitivity and affects survival of cervical cancer patients. Mol Cancer Ther 2008; 7: 1376-1385.

37 Maa MC, Lee JC, Chen YJ, Chen YJ, Lee YC, Wang ST et al. Eps8 facilitates cellular growth and motility of colon cancer cells by increasing the expression and activity of focal adhesion kinase. J Biol Chem 2007; 282: 19399-19409.

38 Chu PY, Liou JH, Lin YM, Chen CJ, Chen MK, Lin SH et al. Expression of Eps8 correlates with poor survival in oral squamous cell carcinoma. Asia Pac J Clin Oncol 2012; 8: e77-81.

39 Welsch T, Endlich K, Giese T, Buchler MW, Schmidt J. Eps8 is increased in pancreatic cancer and required for dynamic actin-based cell protrusions and intercellular cytoskeletal organization. Cancer Lett 2007; 255: 205-218.

40 Kang H, Wilson CS, Harvey RC, Chen I-M, Murphy MH, Atlas SR et al. Gene expression profiles predictive of outcome and age in infant acute lymphoblastic leukemia: a Children’s Oncology Group study. Blood 2012; 119.

41 Robert E. Hewitt, Andrew McMarlin, David Kleiner, Robert Wersto, Patrick Martin, Maria Tsoskas et al. Validation of a model of colon cancer progression. Journal of Pathology 2000; 192: 446-454.
42 Betel D, Wilson M, Gabow A, Marks DS, Sander C. The microRNA.org resource: targets and expression. Nucleic Acids Res 2008; 36: D149-153.

43 Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 2005; 120: 15-20.

44 Krek A, Grun D, Poy MN, Wolf R, Rosenberg L, Epstein EJ et al. Combinatorial microRNA target predictions. Nat Genet 2005; 37: 495-500.

45 Rhodes DR, Yu J, z S, Deshpandez N, Varambally R, Ghosh D et al. ONCOMINE: A Cancer Microarray Database and Integrated Data-Mining Platform. Neoplasia 2004; 6.

46 Cekaite L, Rantala JK, Bruun J, Guriby M, Ågesen TH, Danielsen SA et al. MiR-9, -31, and -182 Deregulation Promote Proliferation and Tumor Cell Survival in Colon Cancer. Neoplasia 2012; 14: 868-IN821.

47 Inoue A, Yamamoto H, Uemura M, Nishimura J, Hata T, Takemasa I et al. MicroRNA-29b is a Novel Prognostic Marker in Colorectal Cancer. Ann Surg Oncol 2015; 22 Suppl 3: S1410-1418.

48 Lijun Xu, Yue Zhang, Hui Wang, Guanhua Zhang, Yanqing Ding, Zhao L. Tumor suppressor miR-1 restrains epithelial-mesenchymal transition and metastasis of colorectal carcinoma via the MAPK and PI3K/AKT pathway. Journal of Translational Medicine 2014; 12.

49 Park YR, Lee ST, Kim SL, Liu YC, Lee MR, Shin JH et al. MicroRNA-9 suppresses cell migration and invasion through downregulation of TM4SF1 in colorectal cancer. International journal of oncology 2016; 48: 2135-2143.

50 Sun P, Sun D, Wang X, Liu T, Ma Z, Duan L. miR-206 is an independent prognostic factor and inhibits tumor invasion and migration in colorectal cancer. Cancer biomarkers : section A of Disease markers 2015; 15: 391-396.

51 Vickers MM, Bar J, Gorn-Hondermann I, Yarom N, Daneshmand M, Hanson JE et al. Stage-dependent differential expression of microRNAs in colorectal cancer: potential role as markers of metastatic disease. Clin Exp Metastasis 2012; 29: 123-132.

52 Xu K, Liu X, Mao X, Xue L, Wang R, Chen L et al. MicroRNA-149 suppresses colorectal cancer cell migration and invasion by directly targeting forkhead box transcription factor FOXM1. Cell Physiol Biochem 2015; 35: 499-515.

53 Shi ZM, Wang XF, Qian X, Tao T, Wang L, Chen QD et al. MiRNA-181b suppresses IGF-1R and functions as a tumor suppressor gene in gliomas. RNA 2013; 19: 552-560.

54 Shin KH, Bae SD, Hong HS, Kim RH, Kang MK, Park NH. miR-181a shows tumor suppressive effect against oral squamous cell carcinoma cells by downregulating K-ras. Biochem Biophys Res Commun 2011; 404: 896-902.

55 Wang XF, Shi ZM, Wang XR, Cao L, Wang YY, Zhang JX et al. MiR-181d acts as a tumor suppressor in glioma by targeting K-ras and Bcl-2. J Cancer Res Clin Oncol 2012; 138: 573-584.

56 Kim M, Slack FJ. MicroRNA-mediated regulation of KRAS in cancer. JOURNAL OF HEMATOLOGY & ONCOLOGY 2014; 7.

57 Ma Z, Qiu X, Wang D, Li Y, Zhang B, Yuan T et al. MiR-181a-5p inhibits cell proliferation and migration by targeting Kras in non-small cell lung cancer A549 cells. Acta Biochim Biophys Sin (Shanghai) 2015; 47: 630-638.

58 Hashimoto Y, Akiyama Y, Otsubo T, Shimada S, Yuasa Y. Involvement of epigenetically silenced microRNA-181c in gastric carcinogenesis. Carcinogenesis 2010; 31: 777-784.

59 Zhang Q, Sun H, Jiang Y, Ding L, Wu S, Fang T et al. MicroRNA-181a suppresses mouse granulosa cell proliferation by targeting activin receptor IIA. PLoS One 2013; 8: e59667.

60 Mataki H, Seki N, Chiyomaru T, Enokida H, Goto Y, Kumamoto T et al. Tumor-suppressive microRNA-206 as a dual inhibitor of MET and EGFR oncogenic signaling in lung squamous cell carcinoma. International journal of oncology 2015; 46: 1039-1050.

61 Fu X, Cui Y, Yang S, Xu Y, Zhang Z. MicroRNA-613 inhibited ovarian cancer cell proliferation and invasion by regulating KRAS. Tumour Biol 2016; 37: 6477-6483.

62 Li D, Li DQ, Liu D, Tang XJ. MiR-613 induces cell cycle arrest by targeting CDK4 in non-small cell lung cancer. Cellular oncology (Dordrecht) 2016; 39: 139-147.

63 Chen X, Shi J, Zhong J, Huang Z, Luo X, Huang Y et al. miR-1, regulated by LMP1, suppresses tumour growth and metastasis by targeting K-ras in nasopharyngeal carcinoma. Int J Exp Pathol 2015; 96: 427-432.

64 Hartz JM, Engelmann D, Furst K, Marquardt S, Spitschak A, Goody D et al. Integrated Loss of miR-1/miR-101/miR-204 Discriminates Metastatic from Nonmetastatic Penile Carcinomas and Can Predict Patient Outcome. J Urol 2016.

65 Jin L, Li Y, Liu J, Yang S, Gui Y, Mao X et al. Tumor suppressor miR-149-5p is associated with cellular migration, proliferation and apoptosis in renal cell carcinoma. Molecular medicine reports 2016; 13: 5386-5392.