由早先的研究得知，Eps8 (EGF receptor pathway substrate No.8) 同時為一些receptor tyrosine kinases與 cytoplasmic tyrosine kinase Src 的受質之一。在許多不同的細胞株中，Eps8都有分子量97-kDa 和68-kDa 兩種isoforms。在我們先前的研究指出：(1) 在C3H10T1/2纖維母細胞中過度表達p97Eps8不但能促使細胞focus的形成，在動物實驗中也能促進腫瘤的形成。(2) 在v-Src transformed細胞 (IV5)中，抑制細胞內Eps8的表現，不僅會造成細胞生長下降，在動物實驗中也能抑制腫瘤的形成。因此，Eps8的確在細胞的轉型及腫瘤的形成扮演重要的角色。然而，在v-Ha-Ras transformed細胞中，我們發現p97Eps8與p68Eps8有大量表達的現象。因此，為了探討Eps8在v-Ha-Ras mediated cell growth 及 transformation中所扮演的角色，我們利用RNA interference (RNAi) 的技術，挑出表現p97eps8 and eps8 (both p97 kDa and p68 kDa) small interference RNA (siRNA) -overexpressing的細胞株。在這些細胞株中，我們發現Eps8蛋白質及mRNA的表達有受到抑制的現象，而且細胞的生長不論在培養皿上或在軟洋菜膠內，都會受到明顯的抑制。接著我們以fluorescence activated cell sorting (FACS) 分析細胞的cell cycle，從實驗結果發現大量表達p97eps8 siRNA及eps8 siRNA的細胞株有G1 phase-retarded及S-phase減少的現象，同時發現p97eps8 siRNAs會造成p21 Waf1/Cip1的表現量增加，而抑制Cyclin E的表達；但是eps8 siRNAs則會增加p27Kip1的表達，而抑制Cyclin D1的表現，並且都會使Rb的磷酸化下降。最後，我們也發現當抑制細胞內Eps8蛋白質的表達時，會造成Ras下游的effector proteins，ERK及AKT的活性下降。因此，我們認為Eps8可能透過ERK及AKT的訊息傳遞路徑而抑制p21Waf1/Cip1或p27Kip1的表達，及促進Cyclin D1或Cyclin E大量表現，使得Rb高度磷酸化後無法與E2F結合，於是E2F可以進一步地轉錄細胞週期進行所需之基因，讓細胞能順利進入S phase而促進細胞的生長。

　Eps8 (EGF receptor pathway substrate No.8) is a substrate for both receptor and non-receptor tyrosine kinases. It has two isoforms, p97Eps8 and p68Eps8 in many cell lines. Our previous studies indicated that :
(1) C3H10T1/2 fibroblast overexpressing p97Eps8 not only exhibits the ability of focus formation in cell culture but also promotes the tumor formation in mice; (2) down-regulation of Eps8 inhibits the cellular growth of v-Src transformed cells, and suppresses tumor formation in mice. Therefore, Eps8 plays an important role in v-Src mediated cell transformation and tumor formation. In this study, we observed that Eps8 protein expression was elevated in v-Ha-Ras transformed cells. In order to examine the role of Eps8 in v-Ha-Ras-mediated cell growth and transformation, we generated p97eps8 and eps8 (both p97 kDa and p68 kDa) small interference RNA (siRNA)-overexpressing stable cell lines. In these cell lines, we observed that Eps8 protein and mRNA expression is reduced and the cell growth is significantly slower than the parental v-Ha-Ras transformed cells. FACS analysis indicitated that these Eps8 knockdown cells exhibit retardation of G1 phase progresssion as compared to the control cells. Then, we observed that the induction of p21Waf1/Cip1 or p27Kip1 and reduction of Cyclin D1 or Cyclin E leading to the decrease of Rb hyperphosphorylation that may contribute to the slower progression of G1 phase in Eps8 knockdown cells. In addition, we observed that v-Ha-Ras-mediated activation of ERK and AKT is reduced in these Eps8 knockdown cells. Therefore, Eps8 may participate in v-Ha-Ras mediated cell growth and transformation through ERK and AKT activation to reduce p21Waf1/Cip1 or p27Kip1 expression and to increase Cyclin D1 and Cyclin E expression resulting in Rb hyperphosphorylation that further promotes cell cycle progression.

Adrienne D Cox and Channing J Der (2003) The dark side of Ras: regulation of apoptosis. Oncogene 22, 8999-9006.

Aktas H., H. Cai & G. M. Cooper (1997) Ras links growth factor signaling to the cell cycle machinery via regulation of cyclin D1 and the Cdk inhibitor p27KIP1. Mol Cell Biol 17, 3850-3857.

Almoguera, C., D. Shibata, K. Forrester, J. Martin, N. Arnheim, andM. Perucho. (1988) Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes. Cell 53, 549-554.

Barbacid, M. (1987) ras genes. Annu. Revi. Biochem. 56, 779-827.

Beitel, G. J., S. G. Clark, and H.R. Horvitz. (1990) Caenorhabditis elegant ras gene let-60 acts as a switch in the pathway of vulval induction. Nature 348, 503-509.

Benito, M., A. Porras, A. R. Nebreda, and E. Santos. (1991) Differentiation of 3T3-L1 fibroblasts to adipocytes induced by transfection of ras oncogenes. Science 253, 565-568.

Bernstein E, Causy AA, Hammond SM and Hannon GJ. (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363-366.

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

Bos, J.L.(1989) ras oncogenes in human cancer: a review. Cancer Res 49, 4682-4689.

Daum, G., I. Eisenmann-Tappe, H. W. Fries, J. Troppmair, and U. R. Rapp. (1994) The ins and outs of Raf kinase. TIBS 19, 474-480.

Delong, L., and H. W. Lu. (1994) Oncogenes, protein kinase, and signal transduction. J. Biomed. Sci. 1, 65-82.

Dent, P., W. Haser, T. A. Haystead, L. A. Vincent, T. M. Roberts, and T. W. Sturgill. (1992) Activation of mitogen-activated protein kinase by v-Raf in NIH3T3 cells and in vitro. Science 257, 1404-1407

Elion EA.(1998) Routing MAP kinase cascades. Science 281, 1625-1626

Fanger GR, Gerwins P, Widmann C, Jarpe MB, Johnson GL. (1997) MEKKs, GCKs, MLKs, PAKs, TAKs, and Tpls: upstream regulators of the c-Jun amino-terminal kinases? Curr Opin Genet Dev. 7, 67-74

Fazioli F, Minichiello L, Matoska V, Castagnino P, Miki T, Wong WT and Di Fiore PP. (1993) Eps8, a substrate for the epidermal growth factor receptor kinase, enhances EGF-dependent mitogenic signals. EMBO J. 12, 3799-3808.

Feig L, and G. M. Cooper. (1988) Inhibition of NIH3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP. Mol. Cell. Biol. 8, 3235-3243

Fire EW, Siqun XU, Montgomery MK, Kostas SA, Driver SE and Mello CC. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811.

Garrington TP, Johnson GL. (1999) Organization and regulation of mitogen-activated protein kinase signaling pathways. Curr. Opin. Cell Biol. 11, 211-218

Hammon d SM, Boettcher S, Caudy AA, Kobayashi R and Hannon GJ. (2001) Argonaute 2, a link between genetic and biobhemical analyses of RNAi. Science 293, 1146-1150.

Hanada M, Feng J and Hemmings BA. (2004) Structure, regulation and function of PKB/Akt- a major therapeutic target. Biochim. Biophys. Acta. 1697, 3-16.

Hu Q, A. Klippel, A. Muslin, W. Fantl, and L. T. Walliam. (1995) Ras-dependent induction of cellular response by constitutively active phosphatidylinositol-3-kinase. Science 268, 100-102

Innocenti M, Frittoli E, Ponzanelli I, Falck JR, Bracmann SM, Di Fiore P P (2003) Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8, Abi1, and Sos-1. J. Cell Biol. 160, 17-23.

Julian Downward. (2003) Targeting Ras signaling pathways in cancer therapy. Nature review cancer. 3, 11-13.

Kauffmann-Zeh, Rodriguez-Viciana, Ulruch C. (1997) Suppression of c-Myc-induced apoptosis by Ras signaling through PI3K and PKB. Nature 385, 544-548.

Karlsson T, Songyang Z, Landgren E, Lavergne C, Di Fiore PP, Anafi M, Pawson T, Cantley LC, Claesson-Welsh L and Welsh M. (1995) Molecular interactions of the Src homology 2 domain protein Shb with phosphotyrosine residues, tyrosine kinase receptors and Src homology 3 domain proteins. Oncogene 10, 1475-1483.

Kawada M., S. Yamagoe, Y. Murakami, K. Suzuki, S. Mizuno & Y. Uehara (1997) Induction of p27Kip1 degradation and anchorage independence by Ras through the MAP kinase signaling pathway. Oncogene 15, 629-637.

Kennedy, S. Wagner, S. Conzen, J. Jordan. (1997) The PI3-kinase/Akt signaling pathway delivers an anti-apoptotic signal. Genes Dev. 11, 701-713.

Khosravi-Far, R. and C. Der. (1994) The Ras signal transduction pathway. Cancer Metast. Rev. 13, 67-89.

Kulik G, a. Klippel, and M. J. Weber. (1997) Antiapoptotic signaling by the Insulin-like growth factor1 receptor, phosphatidylinositol 3-Kinase, and Akt. Mol. Cell. Biol. 17, 1595-1606.

Kun Jiang, Jiazhi Sun, Jin Cheng, Julie Y. (2004) Akt mediates Ras downregulation of RhoB, a suppressor of tansformation, invasion, and metastasis. Mol Cell Biol. 24, 5565-5576.

Kyriakis, J. M., H. App, X. F. Zhang, P. Banerjee and J. Arruch. (1992) Raf-1 activates MAP kinase-kinase. Nature 358, 417-421.

Lambert JM, Lambert QT, Reuther GW, Malliri A, Siderovski DP, Sondek J, Collard JG and Der CJ. (2002) Tiam1 mediates Ras activation of Rac by a PI(3)K-independent mechanism. Nat. Cell. Biol. 4, 621-625.

Leu T-H, Yeh H-H, Huang C-C, Chuang Y-C, Su S-L and Maa M-C. (2004) Participation of p97Eps8 in Src-mediated transformation. J Biol Chem. 279, 9875-9881.

Leone G., J. DeGregori, R. Sears, L. Jakoi & J. R. Nevins (1997) Myc and Ras collaborate in inducing accumulation of active cyclin E/Cdk2 and E2F. Nature 387, 422-426.

Lynn B, Gretchen A, Aylin S, Aidan McFall, Hong Zhou. (2004) Involvment of Ras activation in human breast cancer cell signaling, invasion, and anoikis. Cancer research. 64, 4585-4592.

Maa M-C, Lai J-R, Lin R-W and Leu T-H. (1999) Enhancement of tyrosyl phosphorylation and protein expression of eps8 by v-Src. Biochim. Biophys. Acta. 1450, 341-351.

Maa M-C, Hsieh C-Y and Leu T-H. (2001) Overexpression of p97Eps8 leads to cellular transformation: implication of pleckstrin homology domain in p97Eps8-mediated ERK activation. Oncogene 19, 106-112.

Marcos Malumbres and Angel Pellicer. (1998) Ras pathways to cell control and cell transformation. Frontiers in Bioscience 3, 887-912.

Marshall C. J. (1995) Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation, Cell 80, 179-185.

Matoskova B, Wong WT, Salcini AE, Pelicci PG and Di Fiore PP. (1995) Constitutive phosphorylation of eps8 in tumor cell lines: relevance to malignant transformation. Mol Cell Biol. 15, 3805-3812.

Matoskova B, Wong WT, Seki N, Nagase T, Nomura N, Robbins KC and Di Fiore PP. (1996) RN-tre identifies a family of tre-related proteins displaying a novel potential protein binding domain. Oncogene 12, 2563-2571.

McDonald ER and Deiry WS. (2000) Cell cycle control as a basis for cancer drug development. Int. J. Oncol. 16, 871.

McManus MT and Sharp PA., (2002) Gene silencing in mammals by small interfering RNAs. Nat. Rev. Genet. 3, 737-747.

Ohtsubo M, Theodoras AM, Schumacher J, Roberts JM and Pahano M. (1995) Human cyclin E, a nuclear protein essential for the G1-to-S phase transition. Mol. Cell Biol., 15, 2612.

Parrizas M., A. R. Saltiel, and D. Leroith. (1997) Insulin-like growth factor 1 inhibits apoptosis using the phosphatidylinositol-3-kinase and mitogen-activated protein kinase pathways. J. Biol. Chem. 272, 154-161.

Redddy S, J. H, Huang, and W. S. Liao. (1997) Phosphatidylinositol 3-kinase in interleukin 1 signaling. Physical interaction with the interleukin 1 receptor and requirement in NFkappa B and AP-1 activation. J. Biol. Chem. 272, 29167-29173.

Rita Gallo, Claudia Provenzano, Roberta Carbone, and Stefano Alema. (1997) Regulation of the tyrosine kinase substrate Eps8 expression by growyh factors,v-Src and terminal differentiation. Oncogene 15, 1929-1936.

Robison MJ, Cobb MH. (1997) Mitogen-activated protein kinase pathways. Curr. Opin. Cell Biol. 9, 180-186.

Rodriguea-Viciana, Warne P, Dhand R, and Downward J. (1994) Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature 370, 527-532.
Romashkova JA and Makarov SS. (1999) NF-κB is a target of AKT in anti-apoptotic PDGF signaling. Nature 401, 86-90.
Rosen K, Rak J, Jin J, Kerbel RS, Newman MJ and Filmus J. (1998) Downregulation of the pro-apoptotic protein Bak is required for the ras-induced transformation of intestinal epithelial cells. Curr. Biol. 8, 1331-1334.

Satoh, M., Y. Nakafuka and Y. Kazio. (1992) Function of Ras as moleculat switch in signal transduction. J. Biol. Chem. 267, 24149-24152.

Schaeffer HJ, Weber MJ. (1999) Mitogen-activated protein kinases: specific messengers from ubiquitous messengers. Mol. Cell Biol. 19, 2435-2444.

Scita G, Nordstrom J, Carbone R, Tenca P, Giardina G, Gutkind S, Bjarnegard M, Betsholtz C and Di Fiore PP. (1999) Eps8 and E3B1 transduce signals from Ras to Rac. Nature 401, 290-293.

Scita G, Tenca P, Areces LB, Tocchetti A, Frittoli E, Giardina G, Ponzanelli I, Sini P, Innocenti M and Di Fiore PP. (2001) An effector region in Eps8 is responsible for the activation of the Rac-specific GEF activity of Sos-1 and for the proper localization of the Rac-based actin-polymerizing machine. J. Cell Biol. 154, 1031-1044.

Serrano M, Lin AW, McCurrach ME, Beach D. (1997) Oncogenic Ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell 88, 593-602.

Sulciner DJ, Irani K, Yu ZX, Ferrans VJ, Goldschmidt-Clermont P and Finkel T. (1996) Rac1 regulates a cytokine-stimulated, redox-dependent pathway necessary for NF-kappaB activation. Mol. Cell. Biol. 16, 7115-7121.

Takuwa N. & Y. Takuwa (1997) Ras activity late in G1 phase required for p27kip1 downregulation, passage through the restriction point, and entry into S phase in growth factor-stimulated NIH 3T3 fibroblasts. Mol Cell Biol.17, 5348-5358.

Treisman R. (1996) Regulation of transcription by MAP kinase cascades. Curr. Opin. Cell Biol. 8, 205-215.

Vara JA, Casado E, Castro JD, Cejas P, Belda C and Baron M. (2004) PI3K/Akt signaling pathway and cancer. Cancer Treat Rev 30, 193-204.

Vermeulen K, Van Bockstaele DR and Berneman ZN. (2003) The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer. Cell Prolif. 36, 131-149.

Vojtek A, Hollenberg S, and Cooper J. (1993) Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell 74, 205-214.

Wittinghofer A, Herrmann C. (1995) Ras-effector interactions, the problem of specificity. FEBS Lett 369, 52-56

Wong WT, Carlomagno F, Druck T, Barletta C, Croce CM, Huebner K, Kraus MH and Di Fiore PP. (1994) Evolutionary conservation of the eps8 gene and its mapping to human chromosome 12q23-q24. Oncogene 9, 3057-3061.

Zamore PD, Tuschl T, Sharp PA and Bartel DP. (2000) RNAi: Double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101, 25-33.