臺灣博碩士論文加值系統

前列腺癌對於雄性素去除治療法產生耐受性使其在治療上十分棘手，其中，雄性素受體持續活化可促進前列腺癌惡化及擺脫雄性素去除所造成的治療效果，正常狀態下雄性素受體受到雄性素作用調控，但是在雄性素去除治療法失效的情況下，前列腺癌細胞產生許多改變造成雄性素受體可藉由其他方式活化，在目前的研究中HER2被認為是其中一個的可能因子。HER2對於乳癌的惡化以及對tamoxifen治療產生耐受性上扮演重要的角色，然而，在前列腺癌惡化過程中HER2所扮演的角色仍然並不清楚。在此研究中，我們利用帶有PSA-reporter的22Rv1前列腺癌細胞株分析HER2促成前列腺癌惡化的訊息傳遞機制，我們發現HER2的表現量以及活化程度的提升與細胞的生長及細胞群落形成能力上升相關，此外，利用chemical genetic的方式找出PI3K、Rac1、p38以及IKKα受到
HER2 的調控，HER2 藉由PI3K-Rac1-p38-IKKα訊息途徑調控雄性素受體的活
性，另一方面，抑制這個訊息途徑將會造成前列腺癌細胞進行細胞凋亡，除此之
外，p38 的表現量與活化狀態會隨著HER2 表現量改變而同時提升，我們更近一
步的在組織免疫染色上證明HER2 表現量會隨著前列腺癌惡化而提升。綜合我們
的實驗結果，我們發現HER2 影響雄性素受體活性的訊息途徑可能是透過
PI3K-Rac1-p38-IKKα而不是藉由Akt 或Erk1/2。對於HER2 調控雄性素受體訊息
途徑的了解，將有助於提供治療目標，進而治療惡化的前列腺癌或提升治療效果。

Escaping from androgen ablation therapy makes prostate cancer intractable. The androgen receptor (AR) remains active and supportive to the scenario of PCa development. Deregulations of normal constraints on hormone action activate AR in androgen ablation therapy. Among these deregulation mechanisms, HER2 is considered as one factor providing outlaw pathway to activate AR. HER2 is well studied in breast cancer progression and its tamoxifen resistance, but role of HER2 in prostate cancer progression remains unclear. Here we investigated the molecular basis downstream of HER2 signaling regarding prostate cancer progression. In 22Rv1 reporter cell lines, higher growth rate and colony forming ability correlate with increase of expression and activation level of HER2. We also showed that HER2-specific inhibitor AG825 decreases AR-activated transcription. Analysis on signaling molecules downstream of HER2 for involvement in AR-activated transcription revealed a complete inhibition of AR activity by targeting to PI3K, Rac1, p38α/β and IKKα IKKα. Moreover, inhibitions on these proteins decline the growth rate
and colony forming by inducing apoptosis. On the other hand, increase of p38 levels
and activities correlate with the HER2 expression level. Futhermore,
immnohistological staining in clinical tissue array demonstrated HER2 expression
levels alone with prostatic tumor progression stages. Our data suggest that HER2
overexpression promotes PCa progression might through a PI3K-Rac1-p38α/β-IKKα pathway and is independent of Akt and Erk1/2. This new pathway might
provide possible targets for treating prostate cancer or aid in prevention.

中文摘要…………………………………………………………………I
ABSTRACT………………………………………….…………………II
CONTENTS……………………………………………………...…III-IV

INTRODUCTION…………………………………………………......1-3
MATERIALS AND METHODS…………………………………..….4-7
Cells and reagents……………………………………………………...4
Luciferase assays………………………………………………………4
Growth curve and anchorage-dependent colony forming assays……...4
Apoptosis assays……………………………………………………….5
Western blotting……………………………………………………….5
Rac1 activation assay………………………………………………….6
Caspase 3 & 7 activity assay…………………………………………..7
Immunohistochemical staining………………………………………...7
RESULTS…………………………………………………………......8-17
PSA reporter cell lines exhibit different growth rate in normal and
steroid-deficiency media…………………………………………………..…8
HER2-mediated signal correlates to high growth rate and colony forming rate in normal and steroid-deficiency environment……………………..………9
HER2-mediated signaling is essential in AR-mediated PSA reporter activity.10
HER2-mediated signaling regulates AR-mediated PSA reporter activity through PI3K-Rac1-dependent pathway………….………………………..10
Rac1-mediated regulation of androgen-stimulated PSA-luciferase expression through p38 not JNK……………………………………………………….12
Increases of p38 level and activities among 22Rv1 derived reporter cells…....13
Inhibition of HER2 by AG825 decreases Rac1 and p38 activities in PSA103E cells……………………………………………………………………………14
Inhibitions of HER2-mediated signaling pathway decreases cell survival of PSA103E cells mainly through induce apoptosis…………………………..14
Anti-androgens function as antagonist of AR in PSA-reporter cell line……...16
Increases of HER2 expression level in clinical prostatic tumor specimens accompany with progress stages…………………………………………….17
DISCUSSION……………………………..………………………....18-21
FIGURE………………………………………………………………………..22-48
REFERENCE…………………………………………………………………49-52

Abreu-Martin, M.T., Chari, A., Palladino, A.A., Craft, N.A. and Sawyers, C.L. (1999) Mitogen-activated protein kinase kinase kinase 1 activates androgen receptor-dependent transcription and apoptosis in prostate cancer. Mol Cell Biol, 19, 5143-5154.
Bogoyevitch, M.A., Boehm, I., Oakley, A., Ketterman, A.J. and Barr, R.K. (2004) Targeting the JNK MAPK cascade for inhibition: basic science and therapeutic potential. Biochim Biophys Acta, 1697, 89-101.
Bourguignon, L.Y., Zhu, H., Zhou, B., Diedrich, F., Singleton, P.A. and Hung, M.C. (2001) Hyaluronan promotes CD44v3-Vav2 interaction with Grb2-p185(HER2) and induces Rac1 and Ras signaling during ovarian tumor cell migration and growth. J Biol Chem, 276, 48679-48692.
Chen, C.D., Welsbie, D.S., Tran, C., Baek, S.H., Chen, R., Vessella, R., Rosenfeld, M.G. and Sawyers, C.L. (2004) Molecular determinants of resistance to antiandrogen therapy. Nat Med, 10, 33-39.
Craft, N., Shostak, Y., Carey, M. and Sawyers, C.L. (1999) A mechanism for hormone-independent prostate cancer through modulation of androgen receptor signaling by the HER-2/neu tyrosine kinase. Nat Med, 5, 280-285.
Craig, R., Larkin, A., Mingo, A.M., Thuerauf, D.J., Andrews, C., McDonough, P.M. and Glembotski, C.C. (2000) p38 MAPK and NF-kappa B collaborate to induce interleukin-6 gene expression and release. Evidence for a cytoprotective autocrine signaling pathway in a cardiac myocyte model system. J Biol Chem, 275, 23814-23824.
Feldman, B.J. and Feldman, D. (2001) The development of androgen-independent prostate cancer. Nat Rev Cancer, 1, 34-45.
Gao, Y., Dickerson, J.B., Guo, F., Zheng, J. and Zheng, Y. (2004) Rational design and characterization of a Rac GTPase-specific small molecule inhibitor. Proc Natl Acad Sci U S A, 101, 7618-7623.
Gioeli, D., Ficarro, S.B., Kwiek, J.J., Aaronson, D., Hancock, M., Catling, A.D., White, F.M., Christian, R.E., Settlage, R.E., Shabanowitz, J., Hunt, D.F. and Weber, M.J. (2002) Androgen receptor phosphorylation. Regulation and identification of the phosphorylation sites. J Biol Chem, 277, 29304-29314.
Grossmann, M.E., Huang, H. and Tindall, D.J. (2001) Androgen receptor signaling in androgen-refractory prostate cancer. J Natl Cancer Inst, 93, 1687-1697.
Hynes, N.E. and Lane, H.A. (2005) ERBB receptors and cancer: the complexity of targeted inhibitors. Nat Rev Cancer, 5, 341-354.
Isaacs, J.T. and Isaacs, W.B. (2004) Androgen receptor outwits prostate cancer drugs. Nat Med, 10, 26-27.
Je, J.H., Lee, J.Y., Jung, K.J., Sung, B., Go, E.K., Yu, B.P. and Chung, H.Y. (2004) NF-kappaB activation mechanism of 4-hydroxyhexenal via NIK/IKK and p38 MAPK pathway. FEBS Lett, 566, 183-189.
Kobori, M., Yang, Z., Gong, D., Heissmeyer, V., Zhu, H., Jung, Y.K., Gakidis, M.A., Rao, A., Sekine, T., Ikegami, F., Yuan, C. and Yuan, J. (2004) Wedelolactone suppresses LPS-induced caspase-11 expression by directly inhibiting the IKK complex. Cell Death Differ, 11, 123-130.
Lee, H., Jiang, F., Wang, Q., Nicosia, S.V., Yang, J., Su, B. and Bai, W. (2000) MEKK1 activation of human estrogen receptor alpha and stimulation of the agonistic activity of 4-hydroxytamoxifen in endometrial and ovarian cancer cells. Mol Endocrinol, 14, 1882-1896.
Lee, M.S., Igawa, T., Yuan, T.C., Zhang, X.Q., Lin, F.F. and Lin, M.F. (2003) ErbB-2 signaling is involved in regulating PSA secretion in androgen-independent human prostate cancer LNCaP C-81 cells. Oncogene, 22, 781-796.
Lin, H.K., Yeh, S., Kang, H.Y. and Chang, C. (2001) Akt suppresses androgen-induced apoptosis by phosphorylating and inhibiting androgen receptor. Proc Natl Acad Sci U S A, 98, 7200-7205.
Liu, Y., Majumder, S., McCall, W., Sartor, C.I., Mohler, J.L., Gregory, C.W., Earp, H.S. and Whang, Y.E. (2005) Inhibition of HER-2/neu Kinase Impairs Androgen Receptor Recruitment to the Androgen Responsive Enhancer. Cancer Res, 65, 3404-3409.
Marinari, B., Costanzo, A., Viola, A., Michel, F., Mangino, G., Acuto, O., Levrero, M., Piccolella, E. and Tuosto, L. (2002) Vav cooperates with CD28 to induce NF-kappaB activation via a pathway involving Rac-1 and mitogen-activated kinase kinase 1. Eur J Immunol, 32, 447-456.
Mellinghoff, I.K., Vivanco, I., Kwon, A., Tran, C., Wongvipat, J. and Sawyers, C.L. (2004) HER2/neu kinase-dependent modulation of androgen receptor function through effects on DNA binding and stability. Cancer Cell, 6, 517-527.
Nebreda, A.R. and Porras, A. (2000) p38 MAP kinases: beyond the stress response. Trends Biochem Sci, 25, 257-260.
Olayioye, M.A., Neve, R.M., Lane, H.A. and Hynes, N.E. (2000) The ErbB signaling network: receptor heterodimerization in development and cancer. Embo J, 19, 3159-3167.
Poletti, A., Negri-Cesi, P. and Martini, L. (2005) Reflections on the diseases linked to mutations of the androgen receptor. Endocrine, 28, 243-262.
Rocchi, P., Muracciole, X., Fina, F., Mulholland, D.J., Karsenty, G., Palmari, J., Ouafik, L., Bladou, F. and Martin, P.M. (2004) Molecular analysis integrating different pathways associated with androgen-independent progression in LuCaP 23.1 xenograft. Oncogene, 23, 9111-9119.
Saccani, S., Pantano, S. and Natoli, G. (2002) p38-Dependent marking of inflammatory genes for increased NF-kappa B recruitment. Nat Immunol, 3, 69-75.
Sahai, E. and Marshall, C.J. (2002) RHO-GTPases and cancer. Nat Rev Cancer, 2, 133-142.
Schreiber, S.L. and Bernstein, B.E. (2002) Signaling network model of chromatin. Cell, 111, 771-778.
Sebolt-Leopold, J.S. and Herrera, R. (2004) Targeting the mitogen-activated protein kinase cascade to treat cancer. Nat Rev Cancer, 4, 937-947.
Shin, I., Kim, S., Song, H., Kim, H.R. and Moon, A. (2005) H-Ras-specific activation of Rac-MKK3/6-p38 pathway: its critical role in invasion and migration of breast epithelial cells. J Biol Chem, 280, 14675-14683.
Signoretti, S., Montironi, R., Manola, J., Altimari, A., Tam, C., Bubley, G., Balk, S., Thomas, G., Kaplan, I., Hlatky, L., Hahnfeldt, P., Kantoff, P. and Loda, M. (2000) Her-2-neu expression and progression toward androgen independence in human prostate cancer. J Natl Cancer Inst, 92, 1918-1925.
Song, Y.J., Jen, K.Y., Soni, V., Kieff, E. and Cahir-McFarland, E. (2006) IL-1 receptor-associated kinase 1 is critical for latent membrane protein 1-induced p65/RelA serine 536 phosphorylation and NF-kappaB activation. Proc Natl Acad Sci U S A, 103, 2689-2694.
Stanbrough, M., Bubley, G.J., Ross, K., Golub, T.R., Rubin, M.A., Penning, T.M., Febbo, P.G. and Balk, S.P. (2006) Increased expression of genes converting adrenal androgens to testosterone in androgen-independent prostate cancer. Cancer Res, 66, 2815-2825.
Vivanco, I. and Sawyers, C.L. (2002) The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat Rev Cancer, 2, 489-501.
Wen, Y., Hu, M.C., Makino, K., Spohn, B., Bartholomeusz, G., Yan, D.H. and Hung, M.C. (2000) HER-2/neu promotes androgen-independent survival and growth of prostate cancer cells through the Akt pathway. Cancer Res, 60, 6841-6845.
Xu, Q., Karouji, Y., Kobayashi, M., Ihara, S., Konishi, H. and Fukui, Y. (2003) The PI 3-kinase-Rac-p38 MAP kinase pathway is involved in the formation of signet-ring cell carcinoma. Oncogene, 22, 5537-5544.
Yamamoto, Y., Verma, U.N., Prajapati, S., Kwak, Y.T. and Gaynor, R.B. (2003) Histone H3 phosphorylation by IKK-alpha is critical for cytokine-induced gene expression. Nature, 423, 655-659.
Yang, C., Liu, Y., Lemmon, M.A. and Kazanietz, M.G. (2006) Essential role for Rac in heregulin beta1 mitogenic signaling: a mechanism that involves epidermal growth factor receptor and is independent of ErbB4. Mol Cell Biol, 26, 831-842.
Yang, L., Dan, H.C., Sun, M., Liu, Q., Sun, X.M., Feldman, R.I., Hamilton, A.D., Polokoff, M., Nicosia, S.V., Herlyn, M., Sebti, S.M. and Cheng, J.Q. (2004) Akt/protein kinase B signaling inhibitor-2, a selective small molecule inhibitor of Akt signaling with antitumor activity in cancer cells overexpressing Akt. Cancer Res, 64, 4394-4399.
Yarden, Y. and Sliwkowski, M.X. (2001) Untangling the ErbB signalling network. Nat Rev Mol Cell Biol, 2, 127-137.
Yeh, S., Lin, H.K., Kang, H.Y., Thin, T.H., Lin, M.F. and Chang, C. (1999) From HER2/Neu signal cascade to androgen receptor and its coactivators: a novel pathway by induction of androgen target genes through MAP kinase in prostate cancer cells. Proc Natl Acad Sci U S A, 96, 5458-5463.
Zegarra-Moro, O.L., Schmidt, L.J., Huang, H. and Tindall, D.J. (2002) Disruption of androgen receptor function inhibits proliferation of androgen-refractory prostate cancer cells. Cancer Res, 62, 1008-1013.