||Investigation of Therapeutic Drugs Targeting Eps8 for Colorectal Cancer Treatment
||Department of Pharmacology
大腸直腸癌是世界第三大常見的癌症。據過去研究指出，Eps8在大腸直腸癌病人檢體當中有高度的表現量而Eps8的表現量又會造成癌症患者預後較差的情形。Eps8為一種致癌蛋白質，與致癌基因Src及細胞癌化蛋白質FAK有關。因此，找出抑制Eps8表達的試劑有助於大腸癌治療藥物的發展。本研究的目的是要探討Eps8抑製劑的分子機制，通過connectivity map，我們選用了11種與Eps8表達相關的藥物進行測試，最終篩選出M9作為後續探討。利用人類大腸直腸癌細胞株HT-29，SW480和SW620進行細胞實驗，從細胞存活試驗中我們證實M9對於細胞增生有顯著的抑制效果。通過RT-PCR我們驗證了M9抑制Eps8基因轉錄的能力。另外，從western blot的結果發現M9具有濃度依賴性及時間依賴性而抑制Eps8、FAK和Src蛋白質的表達與活性。在流式細胞儀分析中我們觀察到M9會導致大腸癌細胞滯留在G1細胞週期中，影響細胞生長。以上的實驗結果指出M9可以作為Eps8抑製劑並能有效抑制癌細胞的生長，對於發展成為大腸癌用藥具有莫大的潛力。
Colorectal cancer is the third most common cancer in the world. It has been reported that Eps8, epidermal growth factor receptor pathway substrate 8, is expressed at elevated levels in colon cancer and high levels of Eps8 results in poor prognosis of cancer patients. Eps8 acts as an oncoprotein in human cancer. Therefore, an agent that attenuates Eps8 expression can be developed to be therapeutic drug for colon cancer. The aim of this study is to investigate the underlying mechanisms of M9, a potential Eps8 inhibitor for the treatment of colon cancer. Through connectivity map, which includes 6100 drug-mediated expression profiles, 11 candidate drugs were shown to have anti-correlated expression patterns of Eps8. Human colon cancer cell lines Ht-29, SW480 and SW620 were used as model in this study. Results from cell viability assay determined that M9, one of the 11 candidate drugs, inhibited cell proliferation of the treated cells. Then we validated that M9 inhibited eps8 gene transcription through RT-PCR. Moreover, results from western blot analysis revealed that M9 suppressed the expression of Eps8, the activity of FAK and Src and the expression of FAK and Src in dose- and time- dependent manner. In addition, in the flow cytometry analysis, M9 resulted in G1 phase arrest in the colon cancer cell lines and affecting cell proliferation. These findings indicate that M9 is an Eps8 inhibitor and can inhibit cancer cell growth, suggesting its repositioning potential for colon cancer treatment.
Abstract in English ii
Abstract in Chinese iii
List of Figures vii
General introduction 1
Colorectal cancer 1
EGFR and the treatment of colorectal cancer 1
Connectivity Map 4
Specific aims 5
Material and Experimental Procedures 6
 K. W. Kinzler and B. Vogelstein, "Cancer-susceptibility genes. Gatekeepers and caretakers," Nature, vol. 386, pp. 761, 763, Apr 24 1997.
 E. R. Fearon and B. Vogelstein, "A genetic model for colorectal tumorigenesis," Cell, vol. 61, pp. 759-67, Jun 1 1990.
 B. Vogelstein, N. Papadopoulos, V. E. Velculescu, S. Zhou, L. A. Diaz, and K. W. Kinzler, "Cancer genome landscapes," science, vol. 339, pp. 1546-1558, 2013.
 C. Bokemeyer, I. Bondarenko, A. Makhson, J. T. Hartmann, J. Aparicio, F. de Braud, et al., "Fluorouracil, leucovorin, and oxaliplatin with and without cetuximab in the first-line treatment of metastatic colorectal cancer," J Clin Oncol, vol. 27, pp. 663-71, Feb 10 2009.
 E. Van Cutsem, C. H. Kohne, E. Hitre, J. Zaluski, C. R. Chang Chien, A. Makhson, et al., "Cetuximab and chemotherapy as initial treatment for metastatic colorectal cancer," N Engl J Med, vol. 360, pp. 1408-17, Apr 2 2009.
 Y. Yarden and M. X. Sliwkowski, "Untangling the ErbB signalling network," Nat Rev Mol Cell Biol, vol. 2, pp. 127-37, Feb 2001.
 M. C. Maa, C. Y. Hsieh, and T. H. Leu, "Overexpression of p97Eps8 leads to cellular transformation: implication of pleckstrin homology domain in p97Eps8-mediated ERK activation," Oncogene, vol. 20, pp. 106-12, Jan 4 2001.
 P. P. Di Fiore and G. Scita, "Eps8 in the midst of GTPases," Int J Biochem Cell Biol, vol. 34, pp. 1178-83, Oct 2002.
 P. S. Liu, T. H. Jong, M. C. Maa, and T. H. Leu, "The interplay between Eps8 and IRSp53 contributes to Src-mediated transformation," Oncogene, vol. 29, pp. 3977-89, Jul 8 2010.
 M. C. Maa, J. R. Lai, R. W. Lin, and T. H. Leu, "Enhancement of tyrosyl phosphorylation and protein expression of eps8 by v-Src," Biochim Biophys Acta, vol. 1450, pp. 341-51, Jul 8 1999.
 M.-C. Maa and T.-H. Leu, EPS8, an adaptor protein acts as an oncoprotein in human cancer: INTECH Open Access Publisher, 2013.
 W. Wang, J. B. Wyckoff, V. C. Frohlich, Y. Oleynikov, S. Huttelmaier, J. Zavadil, et al., "Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling," Cancer Res, vol. 62, pp. 6278-88, Nov 1 2002.
 Y. J. Chen, M. R. Shen, Y. J. Chen, M. C. Maa, and T. H. Leu, "Eps8 decreases chemosensitivity and affects survival of cervical cancer patients," Mol Cancer Ther, vol. 7, pp. 1376-85, Jun 2008.
 M.-C. Maa, J.-C. Lee, Y.-J. Chen, Y.-J. Chen, Y.-C. Lee, S.-T. Wang, et al., "Eps8 facilitates cellular growth and motility of colon cancer cells by increasing the expression and activity of focal adhesion kinase," Journal of Biological Chemistry, vol. 282, pp. 19399-19409, 2007.
 P. Y. Chu, J. H. Liou, Y. M. Lin, C. J. Chen, M. K. Chen, S. H. Lin, et al., "Expression of Eps8 correlates with poor survival in oral squamous cell carcinoma," Asia Pac J Clin Oncol, vol. 8, pp. e77-81, Dec 2012.
 Y. Huang, M. Prasad, W. J. Lemon, H. Hampel, F. A. Wright, K. Kornacker, et al., "Gene expression in papillary thyroid carcinoma reveals highly consistent profiles," Proc Natl Acad Sci U S A, vol. 98, pp. 15044-9, Dec 18 2001.
 M. Xu, L. Shorts-Cary, A. J. Knox, B. Kleinsmidt-DeMasters, K. Lillehei, and M. E. Wierman, "Epidermal growth factor receptor pathway substrate 8 is overexpressed in human pituitary tumors: role in proliferation and survival," Endocrinology, vol. 150, pp. 2064-71, May 2009.
 T. Welsch, K. Endlich, T. Giese, M. W. Buchler, and J. Schmidt, "Eps8 is increased in pancreatic cancer and required for dynamic actin-based cell protrusions and intercellular cytoskeletal organization," Cancer Lett, vol. 255, pp. 205-18, Oct 8 2007.
 H. Kang, C. S. Wilson, R. C. Harvey, I. M. Chen, M. H. Murphy, S. R. Atlas, et al., "Gene expression profiles predictive of outcome and age in infant acute lymphoblastic leukemia: a Children's Oncology Group study," Blood, vol. 119, pp. 1872-81, Feb 23 2012.
 M. D. Schaller, C. A. Borgman, and J. T. Parsons, "Autonomous expression of a noncatalytic domain of the focal adhesion-associated protein tyrosine kinase pp125FAK," Mol Cell Biol, vol. 13, pp. 785-91, Feb 1993.
 J. T. Parsons, "Focal adhesion kinase: the first ten years," J Cell Sci, vol. 116, pp. 1409-16, Apr 15 2003.
 S. K. Hanks, L. Ryzhova, N. Y. Shin, and J. Brabek, "Focal adhesion kinase signaling activities and their implications in the control of cell survival and motility," Front Biosci, vol. 8, pp. d982-96, May 1 2003.
 J. Lamb, E. D. Crawford, D. Peck, J. W. Modell, I. C. Blat, M. J. Wrobel, et al., "The Connectivity Map: using gene-expression signatures to connect small molecules, genes, and disease," Science, vol. 313, pp. 1929-35, Sep 29 2006.
 D. Ahmed, P. W. Eide, I. A. Eilertsen, S. A. Danielsen, M. Eknaes, M. Hektoen, et al., "Epigenetic and genetic features of 24 colon cancer cell lines," Oncogenesis, vol. 2, p. e71, 2013.
 J. L. Bos, E. R. Fearon, S. R. Hamilton, M. Verlaan-de Vries, J. H. van Boom, A. J. van der Eb, et al., "Prevalence of ras gene mutations in human colorectal cancers," Nature, vol. 327, pp. 293-7, May 28-Jun 3 1987.
 A. Lievre, J. B. Bachet, D. Le Corre, V. Boige, B. Landi, J. F. Emile, et al., "KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer," Cancer Res, vol. 66, pp. 3992-5, Apr 15 2006.
 A. W. Wyke, M. C. Frame, D. A. Gillespie, A. Chudleigh, and J. A. Wyke, "Mitogenesis by v-Src: fluctuations throughout G1 of classical immediate early AP-1 and mitogen-activated protein kinase responses that parallel the need for the oncoprotein," Cell Growth Differ, vol. 6, pp. 1225-34, Oct 1995.