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And Hedgehog-Signaling Pathways in Prostate Cancer Targeting Estrogen- and Hedgehog-Signaling Pathways in Prostate Cancer _______________________________________________ A Dissertation presented to The Faculty of the Graduate School University of Missouri – Columbia _______________________________________________ In Partial Fulfillment Of the Requirement for the Degree Doctor of Philosophy _______________________________________________ by ANNA ŚLUSARZ Dr. Dennis B. Lubahn, Dissertation Supervisor DECEMBER 2009 © Copyright by Anna Ślusarz 2009 All Rights Reserved The undersigned, appointed by the Dean of the Graduate School, have examined the dissertation entitled Targeting Estrogen- and Hedgehog-Signaling Pathways in Prostate Cancer Presented by Anna Ślusarz A candidate for the degree of Doctor of Philosophy And hereby certify that in their opinion it is worthy of acceptance. Dr. Dennis Lubahn __________________________________________________ Dr. Cynthia Besch-Williford __________________________________________ Dr. William Folk ___________________________________________________ Dr. Venkataseshu Ganjam ____________________________________________ Dr. Mark Martin ____________________________________________________ ACKNOWLEDGMENTS I would like to thank my research advisor, Dr. Dennis Lubahn for his patience, encouragement and support throughout the development and completion of this research project. My thanks to my committee members: Drs. Cynthia Besch-Williford, William Folk, Venkataseshu Ganjam and Mark Martin for their advice, guidance and mentoring throughout the course of my studies. I would also like to thank Dr. Warren Zahler, who introduced me to the world of biochemistry and recruited me to the Department of Biochemistry. I owe special thanks to Yi Zhuang, a former student in Dr. Lubahn’s lab for taking me under his wings and introducing me to the work in the lab. I would like to thank Dr. Mary Sakla, Dr. Nader Shenouda, Dr. Pete Ansell, Dr. Wei Zhou, Dr. Jinghua Liu, Dr. Rachel Ruhlen, Dr. Rossy Moo Puc and Leslie Newton, former lab members in the Lubahn lab, who gave me help and support in my research. I would like to thank Jennifer Bogener, our lab manager, without whom we would be lost. I would like to thank all the undergraduates who I worked with, Amy Sestric, Natalie Abert, Katie and Charlie Parker, Byron Bernabe, Will Fisher, Eric Amos Burks, Kat Lucido, and Angela Walker . I would like to thank the current lab members, Dr. Glenn Jackson, Lu Yuan, and Nicholas Starkey for a great collaboration and interesting and stimulating discussion. I would also like to thank Sara Drenkhahn for her friendship and contributions to this project. iii Lastly, I would like to thank my husband, for his support and love. I would also like to express my endless gratitude to my mother, without whom I would not be able to complete my Ph.D. work. Thank you for taking care of our little girls and us in the last couple of years. And I also need to thank my father, who took part in our lives even though he was across the ocean. I would not like to miss thanking my two little princesses, Sonia and Helena, who brought so much happiness into our lives, and who make all this work worth doing. iv TABLE OF CONTENTS ACKNOWLEDGEMENTS……………………………..………….……….…...……....iii LIST OF TABLES …………………………………………………………….…….....viii LIST OF FIGURES ……………………………………………………….………...…...ix LIST OF ABBREVIATIONS ……………………………………………….....……… xii ABSTRACT…………………………………………………………………………...xviii CHAPTER I – BACKGROUND Prostate Cancer……………………………………………………….………...…1 Estrogen Signaling Pathway..…………………...……..……………………...…..2 Estrogen Receptor Mouse Models……………………………………...…………3 Estrogens and Breast Cancer………………………………………………...……4 Estrogens and Prostate Cancer…………………………………………………….5 Stromal - epithelial cell communication in the prostate……………………….….7 Androgens in the prostate………………………………...……………….………7 Anatomy of the prostate……………………………………………………...……9 TRAMP Mouse Model………………………………………………………..…10 Poorly Differentiated Carcinoma (PDC) in TRAMP mice…………………...….11 Cancer Stem Cells………………………………………………......................…12 Botanicals and Cancer……………………………………….………………...…13 Hedgehog Signaling Pathway……………………………………………………20 Hedgehog Signaling and Prostate Cancer……………………………...…...……23 General Overview……………………………………………...………………...24 v CHAPTER II – GENISTEIN REDUCES INCIDENCE OF PROSTATE CANCER IN TRAMP MICE THROUGH ESTROGEN RECEPTOR DEPENDENT MECHANISM Overview……………………………………………………………………....…35 Introduction………………………………………………………………………36 Materials and Methods………………………………………………………..….37 Results……………………………………………………………………………41 Discussion………………………………………………………………….…….45 CHAPTER III – COMMON BOTANICAL COMPOUNDS INHIBIT THE HEDGEHOG SIGNALING PATHWAY IN PROSTATE CANCER Overview………………………………………………………..………………..65 Introduction………………………………………………………………………67 Materials and Methods………………………………………………………..….69 Results……………………………………………………………………………75 Discussion………………………………………………………………….…….79 CHAPTER IV – CROSSTALK BETWEEN ESTROGEN- AND HEDGEHOG SIGNALING PATHWAYS Overview………………………………………………………..………………106 Introduction………………………………………………………………..……107 Materials and Methods………………………….……………………………....108 Results………………………………………………………………………..…111 Discussion………………………………………………………………...…….114 CHAPTER V – FUTURE STUDIES…………………………………………………..129 vi APPENDIX - SPINACH EXTRACTS IN PROSTATE CANCER TREATMENT Overview……………………………………………………………………..…133 Introduction………………………………………………………………..……134 Materials and Methods………………………………………………………….135 Results and Discussion…………………………………………………………135 REFERENCES…………………………………………………………………...…… 143 VITA……………………………………………………………………………………173 vii LIST OF TABLES Table II-1: Effect of diet and genotype on the incidence of prostate cancer in 5 month old TRAMP mice…………………………………………………………………………….48 Table II-2: Effect of diet and genotype on the incidence of prostate cancer in 5 month old TRAMP mice…………………………………………………………………………….50 Table II-3: Tumor incidence for TRAMP mice split by separate study…………………51 Table II-4: Body and organ weights of mice from different dietary groups……………..58 Table III-1: Botanical compounds inhibit Hedgehog pathway activity in TRAMP-C2 cells………………………………………………………………………………………90 Table III-2: Incidence of prostate tumorigenesis in TRAMP mice fed various botanical compounds……………………………………………………………………………….91 Table III-3: Incidence of prostate tumorigenesis in TRAMP mice fed various botanical compounds for each tumor stage………………………………………………...………92 Table A-I: Preliminary results from TRAMP spinach study………………………..….142 viii LIST OF FIGURES Figure I-1: Exon structure, primary transcript, and common mRNA splice variants of the ER………………………………………………………………………………………..26 Figure I-2: Models of estrogen action adapted from Deroo & Korach 2006 …………....28 Figure I-3: Differential ER structure and coactivator recruitment by ER agonists, antagonists, and SERMs…………………………………………………........................29 Figure I-4: Model for prostate cancer progression in TRAMP mice…………………….30 Figure I-5: Histological classification of prostate tumor progression in the ERαKO TRAMP mouse………………...………………………………...………………………31 Figure I-6: Neuroendocrine-like tumor…………………..…………………………..…..32 Figure I -7: The Hedgehog pathway in vertrebrates…………………………………..…33 Figure I-8: Conserved hedgehog pathway…………………………………………….…34 Figure II-1: Mouse body weight from all studies combined……………………………..59 Figure II-2: TRAMP mouse urogenital tract (A) and testes (B) weights………………..60 Figure II-3: Weights for 5 month old TRAMP mouse prostates ………………………..61 Figure II-4: Immunohistochemistry for T-antigen, neuron-endocrine markers, and sex- steroid receptors in TRAMP prostates…………………………………………….……..62 Figure II-5: Effects of Genistein, E2 and ICI on prostate cancer cell growth…………...63 Figure II-6: 5α-androstane-3β,17β-diol in the prostate…………………………………..64 Figure III-1: TRAMP mice express Gli transcription factors……………………………88 Figure III-2: TRAMP-C2 cells express Hedgehog pathway components……………….89 Figure III-3: Botanical compounds inhibit TRAMP-C2 cell growth………………...…..94 ix Figure III-4: Botanical compounds inhibit Hedgehog pathway activity in TRAMP-C2 cells………………………………………………………………………………………95 Figure III-5: Botanical compounds decrease Gli1 protein in TRAMP-C2 cells………...96 Figure III-6: Gli1 mRNA in TRAMP-C2 cells by real-time RT-PCR after time course treatments with cyclopamine and genistein………………………………...……………97 Figure III-7: Shh can stimulate Gli1 mRNA concentrations in TRAMP-C2 cells, genistein can inhibit the stimulated Gli1 expression……………………………………………….98 Figure III-8: Botanical compounds inhibit Shh-stimulated Gli-responsive promoter in Shh Light II cell line………………………………………………………………………….99 Figure III-9: Genistein inhibits cell growth and Hedgehog pathway activity in PC3….100 Figure III-10: Structures on botanical compounds used in mouse diets………………..102 Figure III-11: Botanicals act synergistically to inhibit Gli1 mRNA in TRAMP-C2 …..103 Figure III-12: Total body weights of mice at the end of study…………………………104 Figure III-13: Gli1 protein expression varies in prostates of TRAMP mice…………...105 Figure IV-I: TRAMP-C2 and Shh Light II cells express both estrogen receptors…..…119 Figure IV-2: Oxysterols as novel hedgehog pathway stimulators……………………...120 Figure IV-3: Selected oxysterols stimulate Gli1 mRNA in TRAMP-C2 cells…………121 Figure IV-4: Genistein inhibits stimulation by 25- and 27OHC in TRAMP-C2 cells....122 Figure IV-5: Estrogens and antiestrogens inhibit Gli1 mRNA in TRAMP-C2 cells…..123 Figure IV-6: E2 inhibits Gli1 protein in TRAMP-C2 cells……………………….........124 Figure IV-7: GliBS-reporter
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