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Evaluation of Human Prolactin, Its Antagonist

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Evaluation of Human Prolactin, Its Antagonist Clemson University TigerPrints All Dissertations Dissertations 12-2007 EVALUATION OF HUMAN PROLACTIN, ITS ANTAGONIST, AND ANTAGONIST-BASED FUSION PROTEINS AS CHEMOPREVENTATIVE AND THERAPEUTIC AGENTS Seth Tomblyn Clemson University, [email protected] Follow this and additional works at: https://tigerprints.clemson.edu/all_dissertations Part of the Oncology Commons Recommended Citation Tomblyn, Seth, "EVALUATION OF HUMAN PROLACTIN, ITS ANTAGONIST, AND ANTAGONIST-BASED FUSION PROTEINS AS CHEMOPREVENTATIVE AND THERAPEUTIC AGENTS" (2007). All Dissertations. 171. https://tigerprints.clemson.edu/all_dissertations/171 This Dissertation is brought to you for free and open access by the Dissertations at TigerPrints. It has been accepted for inclusion in All Dissertations by an authorized administrator of TigerPrints. For more information, please contact [email protected]. EVALUATION OF HUMAN PROLACTIN, ITS ANTAGONIST, AND ANTAGONIST-BASED FUSION PROTEINS AS CHEMOPREVENTATIVE AND THERAPEUTIC AGENTS __________________________________________________________________ A Dissertation Presented to the Graduate School of Clemson University __________________________________________________________________ In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Microbiology __________________________________________________________________ by Seth Tomblyn December 2007 __________________________________________________________________ Accepted by: Wen Y Chen, PhD, Committee Chair Charles D Rice, PhD Thomas R Scott, PhD Lesly A Temesvari, PhD ABSTRACT Cancer is a collection of diseases with many different manifestations and is the second leading cause of death in the United States. Breast cancer accounts for nearly one third of cancer diagnosis in women. Prolactin (PRL) functions as a lactogen and as a mammary gland differentiation factor. PRL acts in an autocrine/paracrine manner within the mammary gland and in breast tumors which implies PRL may be involved in breast cancer progression. This is corroborated by the PRLR over-expression in breast cancer cells lines and the majority of patient biopsies. These reasons make PRL and PRLR attractive targets for breast cancer treatment and prevention. Transgenic mice expressing hPRL or G129R, under the regulation of the metallothionein (Mt) promoter, were fed a chemical carcinogen, 9,10-Dimethyl-1,2-benzanthracene (DMBA). G129R transgenic mice exhibited decreased growth rate of chemically-induced tumors, while hPRL transgenic mice had an increased cancer rate. Microarray analysis revealed that hPRL transgenic mammary gland showed an expression pattern similar to those of a pregnant mouse, while the G129R transgenic gland revealed an increase in various apoptotic markers. Previously, fusion proteins composed of a PRLR antagonist (G129R) and anti-tumor domains were developed; these included fusions with an angiogenesis inhibitor (Endostatin), an immune system modulator (interleukin-2), and a cytotoxin (PE38KDEL). The rationale was that each fusion protein would target the mammary gland via the G129R moiety and attack different hallmarks common to tumor cells via the second moiety. A novel clinically-relevant model was generated by surgically removing spontaneous mammary tumors from MMTV-neu transgenic mice and monitoring tumor recurrence while treating with the fusion protein cocktail. Tumor recurrence was significantly delayed in groups treated with the fusion proteins in comparison to the control group. In conclusion, targeting multiple hallmarks of cancer using a combination of dual function therapeutics was highly effective in the aggressive MMTV-neu mouse tumor model. DEDICATION To the only two loves of my life, my wife, Chrystal, and my daughter, Elizabeth. iii ACKNOWLEDGMENTS I would like to thank all the people that have made this work possible. Dr. Wen Chen for his patience and guidance, he has helped me acquire knowledge in a completely new field of study, cancer research, and taught me critical skills that will aid in my development as a scientist. I would also like to thank my committee members, Dr. Charles Rice, Dr. Thomas Scott, and Dr. Lesly A Temesvari for their helpful comments and guidance. I would also like to thank my colleagues and friends: Jang Pyo Park, Dr. Alison Springs, Dr. Karl Franek, Dr. Susan Peirce, Dr. Michele Scotti, Dr. Isabelle Jacquemart, Dr. Lori and Eric Holle, Guillermo Deangulo, Keneisha Burrell, and Dr. Thomas Wagner. I would like extend special thanks to John F. Langenheim, without your conversations and the competitive ribbing through this experience, I believe this process would have taken much longer. I would like to extend my sincerest appreciation to the Endowment Fund of the Greenville Hospital System and grants from the NIH/NCI (CA105479) and The Susan G. Komen Foundation (BCTR0402985 and BCTR091306) for their financial support. Special thanks are also extended to Faye Bryson and Pathology Consultants of Greenville, SC for their time in preparing and the materials for the labeling of the IHC(P) sections with CD4 and CD31. I would also like to recognize my family and friends from my home in West Virginia as well as my friends from the Upstate. Their motivation has kept my moral up and pushed me to complete this work. iv TABLE OF CONTENTS Title Page...........................................................................................................................................i Abstract.............................................................................................................................................ii Dedication........................................................................................................................................ iii Acknowledgments ...........................................................................................................................iv List of Tables ................................................................................................................................. viii List of Figures ..................................................................................................................................ix List of Abbreviations .........................................................................................................................x 1.0 Introduction................................................................................................................................ 1 1.1 Cancer................................................................................................................................... 2 1.2 Hallmarks of Cancer.............................................................................................................. 2 1.3 Chemotherapy....................................................................................................................... 7 1.3.1 Targeted Therapy...................................................................................................... 8 1.3.1.1 Modulating Ligand Concentration........................................................................... 8 1.3.1.2 Attacking Over-Expressed Receptors .................................................................... 9 1.3.1.3 Modulation of Receptor Signaling ........................................................................ 11 1.3.1.4 Utilizing Targeted Therapies as Delivery Mechanisms ........................................ 12 1.3.2 Combination Therapy ...................................................................................................14 1.3.2.1 Using the Old with the New .................................................................................. 14 1.3.2.2 Targeting the Same Oncogene ............................................................................ 15 1.3.2.3 Hitting the Oncogene Along Its Signaling Cascade ............................................. 16 1.3.2.4 Targeting Multiple Hallmarks of Cancer ............................................................... 18 1.4 Breast Cancer ..................................................................................................................... 19 1.4.1 Etiology .........................................................................................................................19 1.4.2 Classification.................................................................................................................21 1.4.3 Prevention.....................................................................................................................22 1.5 Prolactin .............................................................................................................................. 26 v 1.6 DMBA .................................................................................................................................. 32 1.6.1 Phase I Reactions.........................................................................................................33 1.6.2 Phase II Reactions........................................................................................................34 1.6.3 DNA Adduct Formation.................................................................................................36 1.6.4 Mammary Tumor Formation .........................................................................................37 1.6.5 Alterations of Gene Expression ....................................................................................38 1.7 Mouse Models of Cancer
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