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Ph.D. Dissertation PDK-1/AKT PATHWAY AS TARGETS FOR CHEMOSENSITIZING EFFECTS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Ping-Hui Tseng, M.S. ***** The Ohio State University 2005 Dissertation Committee: Approved by Professor Ching-Shih Chen, Advisor Professor Pui-Kai Li Adviser Graduate Program in Pharmacy Professor Charles L. Shapiro Professor Tatiana M. Oberyszyn ABSTRACT Cancer is the leading causes of human deaths all over the world. The growing understanding in cancer biology provides the researchers to develop molecularly targeted therapeutic strategies. However, the toxicity and resistance limit the applications for molecularly targeted agents. Here, we proposed that inhibiting PI3K/PDK-1/Akt signaling pathway, which is critical in controlling cell survival and proliferation, is able to enhance the therapeutic effects and overcome resistance of the current used molecularly targeted drugs. Imatinib (STI571; Gleevec), a selective bcr-abl tyrosine kinase inhibitor, is approved to treat patients with chronic myelogenous leukemia (CML). However, patients in more advanced phases of CML frequently develop resistance to the treatment. Mutations within the kinase domain for the binding of imatininb attributes as one of the major imatinib-resistant mechanisms. The effects of imatinib each alone and in combination with OSU-03012, a novel celecoxib-derived PDK-1 inhibitor, was evaluated in a panel of Bcr-Abl positive Ba/F3 cell lines without or with mutations. The IC50 values for OSU- 03012 alone were comparable, while the sensitivities to imatinib alone were differed. The combination treatment, however, caused a synergistic enhancement of apoptosis in these cells, including those that are resistant to imatinib. ii HER2/neu is frequently over-expressed in breast cancers that are characterized by aggressive tumor progression and resistant to current therapies. The effects of trastuzumab (Herceptin), a monoclonal antibody targeting to HER2/neu, alone and in combination with OSU-03012 were evaluated in a panel of breast cancer cell lines. The IC50 values for OSU-03012 alone in four cell lines were comparable, while the sensitivities to trastuzumab alone were mainly dependent on the HER2/neu expression level or resistant phenotypes. The combination treatments, however, caused a synergistic enhancement of anti-proliferation in HER2/neu positive cell lines, including SKBR3/IGF-IR, as a resistant phenotype, that responded poorly to trastuzumab. These results demonstrate the potential clinical value of a therapeutic strategy to sensitize cancer cells to molecularly targeted drugs by co-targeting PDK-1/Akt signaling. More importantly, the co-treatments are able to overcome drug resistance. iii Dedicated to my lovely parents iv ACKNOWLEDGMENTS I would like to acknowledge my advisor, Dr. Ching-Shih Chen, for his guidance, encouragement, and providing me such an opportunity to study in OSU. I would also like to express my sincere appreciation to my advisory committee members, Dr. Pui-Kai Li, Dr. Charles L. Shapiro and Dr. Tatiana M. Oberyszyn, for all their help and advice in my research. Moreover, I would like to thank Dr. Mike Zhu, Dr. Druker, and Dr. Pollak for their advice in my research and kindly providing materials. I also want to mention the colleagues in Chen’s Lab, Dr. Kulp, Dr. Song, Dr. Johnson, Dr. Wang, Ya-Ting, Julie, Qiang, Ho-Pi, Chung-Wai, Yvette, Arthur, Kuen- Feng, Chang-Shi, Jim, Leo, Jack and Nicole. Without their involvement in scientific discussion, and technique support, my graduate study won’t be so smooth. Of course, I need to thank Pang, Mei-I, Ya-Wen, Raymond, Jason, and all other friends in Columbus. Because of them, the life outside the graduate study is also full of happiness. v VITA Sep. 19, 1975 ………………………...Born – Taichung, Taiwan 1997 ………………………………….B.S. Chemistry National Taiwan University, Taiwan 1999 ……………………………….M.S. Chemistry National Taiwan University, Taiwan 1999 – 2000. Research Associate Institute of Biomedical Science Academia Sinica, Taiwan 2000 – 2001. Research Associate Institute of Biomedical Science Academia Sinica, Taiwan 2001 – Present ……………………….Research Associate College of Pharmacy, The Ohio State University PUBLICATIONS Research Publications 1. Song, X., Lin, H. P., Johnson, A. J., Tseng, P. H., Yang, Y. T., Kulp, S. K., and Chen, C. S. Cyclooxygenase-2, player or spectator in cyclooxygenase-2 inhibitor- induced apoptosis in prostate cancer cells. J Natl Cancer Inst, 94: 585-591, 2002. 2. Yang, C. C., Lin, H. P., Chen, C. S., Yang, Y. T., Tseng, P. H., Rangnekar, V. M., and Chen, C. S. Bcl-xL mediates a survival mechanism independent of the phosphoinositide 3-kinase/Akt pathway in prostate cancer cells. J Biol Chem, 278: 25872-25878, 2003. vi 3. Kulp, S. K., Yang, Y. T., Hung, C. C., Chen, K. F., Lai, J. P., Tseng, P. H., Fowble, J. W., Ward, P. J., and Chen, C. S. 3-phosphoinositide-dependent protein kinase-1/Akt signaling represents a major cyclooxygenase-2-independent target for celecoxib in prostate cancer cells. Cancer Res, 64: 1444-1451, 2004. 4. Zhu, J., Huang, J. W., Tseng, P. H., Yang, Y. T., Fowble, J., Shiau, C. W., Shaw, Y. J., Kulp, S. K., and Chen, C. S. From the cyclooxygenase-2 inhibitor celecoxib to a novel class of 3-phosphoinositide-dependent protein kinase-1 inhibitors. Cancer Res, 64: 4309-4318, 2004. 5. Tseng, P. H., Lin, H. P., Hu, H., Wang, C., Zhu, M. X., and Chen, C. S. The canonical transient receptor potential 6 channel as a putative phosphatidylinositol 3,4,5-trisphosphate-sensitive calcium entry system. Biochemistry, 43: 11701- 11708, 2004. 6. Lin, H. P., Kulp, S. K., Tseng, P. H., Yang, Y. T., Yang, C. C., Chen, C. S., and Chen, C. S. Growth inhibitory effects of celecoxib in human umbilical vein endothelial cells are mediated through G1 arrest via multiple signaling mechanisms. Mol Cancer Ther, 3: 1671-1680, 2004. 7. Tseng, P. H., Lin, H. P., Zhu, J., Chen, K. F., Hade, E. M., Young, D. C., Byrd, J. C., Grever, M., Johnson, K., Druker, B. J., and Chen, C. S. Synergistic interactions between imatinib mesylate and the novel phosphoinositide-dependent kinase-1 inhibitor OSU-03012 in overcoming imatinib mesylate resistance. Blood, 105: 4021-4027, 2005. FIELDS OF STUDY Major Field: Pharmacy vii TABLE OF CONTENTS Page Abstract. ii Dedication. .iv Acknowledgments . .v Vita . .vi List of Figures. xi List of Abbreviations .. .. xiii Chapters: 1. Introduction…………………………………………………………………………… 1 1.1 Molecular Targeting Cancer Therapy…………………………………………….1 1.2 Phosphoinositide 3 Kinase (PI3K)/Phosphoinositide-dependent Kinase-1 (PDK- 1)/Akt Signaling Pathway ………………………………………………………..3 1.3 PI3K/PDK-1/Akt Pathway in Tumorigenesis ……………………….………….. 5 1.4 Inhibitors of the PI3K/PDK-1/Akt Pathway ………………………….………… 7 1.5 Resistance to Chemotherapeutic Agents ……………………………….………. 10 2. Synergistic Interactions Between Imatinib and The Novel Phosphoinositide-dependent kinase-1 Inhibitor OSU-03012 in Overcoming Imatinib Resistance……………..….19 2.1 Background …………………………………… …………..……………………19 2.1.1 Chronic Myelogenous Leukemia (CML)… ………………………….....…19 2.1.2 Bcr-Abl ...…………………………… …………………………………...22 2.1.3 Imatinib .. …………………………… ……………………………………24 viii 2.1.4 Mechanisms of Imatinib Resistance ………………………………………25 2.2 Experimental Hypothesis …………………………………………. ……………27 2.3 Ba/f3 cell models …………………………………………………. ……………28 2.4 Sensitivity of Different Bcr-Abl Positive Cell Lines to Imatinib ……………….29 2.4.1 Anti-proliferation Effects……………………………………. ……………29 2.4.2 Flow Cytometric Analysis with Annexin V/PI staining …………………..30 2.4.3 Effects of Imatinib Treatment on Akt and Cytochrome c …………………31 2.5 Sensitivity of Different Bcr-Abl Posity Cell Lines to OSU-03012. ….…………32 2.5.1 Anti-proliferation Effects…………………………………………………..32 2.5.2 Flow Cytometric Analysis with Annexin V/PI Staining ………………….33 2.5.3 Immunoblotting …………………………………………………………...33 2.6 OSU-03012 Sensitizes Imatinib-resistant Cells to Imatinib-induced Apoptosis. .34 2.6.1 Anti-proliferation Effects………………………………………………… .34 2.6.2 Flow Cytometric Analysis with Annexin V/PI Staining ………………….34 2.6.3 Combination Index…… …………………………………………………. 35 2.6.4 Effects on Bcr-Abl and Akt………………………………….…………….36 2.7 Discussion ………………………………………………………..……………...37 3. Overcoming Trastuzumab Resistance in HER2/neu-over-expressing Breast Cancer Cells…………….………….………………………………………………………...55 3.1 Background ……………………………………………………………………...55 3.1.1 Breast Cancer …………………...…………………………………………55 3.1.2 HER2/neu in Breast Cancer..……...……………………………………….58 3.1.3 Trastuzumab ……………………………………………………………….60 3.1.4 Mechanisms of Trastuzumab Resistance ………………………………….63 3.2 Experimental Hypothesis …………………………………………………..……65 3.3 In Vitro cell models ………………………………………………………..……66 3.4 Sensitivity of Different HER2/neu Expression Breast Cancer Cell Lines to Trastuzumab ……………………………………………………….……………68 3.4.1 Anti-proliferation Effects ………………………………………………….68 3.4.2 Immunobloting …………………………………………………………….68 3.5 Sensitivity of HER2/neu Expression Breast Cancer Cell Lines to OSU-03012 ...69 3.5.1 Anti-proliferation Effects ………………………………………………….69 3.5.2 Immunobloting …………………………………………………………….70 3.5.3 Immunohistochemical Staining ………………………………………..….71 3.6 OSU-03012 Sensitizes HER2/neu Expressing Cells to Trastuzumab ……..……72 3.6.1 Anti-proliferation effects …………………………………………….,,…..72 3.6.2 Combination Index ……………………………………………………… .72 3.6.3 Immunoblotting …………………………………………………………...73 3.7 Discussion ……………………………………………………………………....74 4. Conclusion
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