NOVEL INSIGHTS INTO BONE MORPHOGENETIC PROTEIN (BMP) AND MAMMALIAN TARGET OF RAPAMYCIN (mTOR) SIGNALING AXIS IN PROSTATE CANCER. by REEMA SAID WAHDAN-ALASWAD Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisor: David Danielpour, Ph. D. Department of Pharmacology CASE WESTERN RESERVE UNIVERSITY August 2011 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of ___Reema Said Wahdan-Alaswad_____ candidate for the _ Ph. D. ______________degree *. (signed)_______Noa Noy, Ph. D.________________ (chair of the committee) _____________David Danielpour, Ph. D. ______________ _____________Bing-Cheng Wang, Ph. D. _____________ _____________Yu-Chung Yang, Ph. D._______________ ____________Johannes von Lintig, Ph. D.______________ ________________________________________________ (date) _December 1, 2010_______________ *We also certify that written approval has been obtained for any proprietary material contained therein. 1 To my parents Said & Urayb, husband Mazen, daughter Lamya, and Siblings Ruba, Rana and Thair. 2 Table of Contents List of Tables .......................................................................................................................5 List of Figures .....................................................................................................................6 Acknowledgements ..........................................................................................................10 Abbreviations ...................................................................................................................12 Abstract .............................................................................................................................15 Chapter 1 Introduction .................................................................................................15 1.1. Prostate Cancer Overview/ Prostate Anatomy ......................................................................... 16 1.1.1 Prostate Zones and Lobes .................................................................................................... 17 1.1.2 Prostate Cancer Etiology .................................................................................................... 17 1.1.3 Prostate Cancer Risk Factors ............................................................................................... 17 1.1.4 Treatment ............................................................................................................................. 18 1.1.5 Prostate Cancer Screening, TNM Staging & Gleason Score ............................................... 19 1.1.6 Progression of Prostate Cancer, Oncogenes, or Tumor Suppressor Genes .......................... 20 1.2. TGF-β superfamily signaling in prostate cancer ....................................................................... 21 1.2.1 The TGF-β superfamily regulatory activity and receptors activation. ................................. 22 1.2.2 Jekyll and Hyde function of TGF-β-Superfamily during prostate carcinogenesis. .............. 23 1.2.3 Alterations of BMP pathway in human cancers .................................................................. 24 1.2.4 Implications of BMP in advanced prostate cancer .............................................................. 26 1.3. Smads: critical mediators of TGF-β signals .............................................................................. 27 1.3.1 Main features of Smad proteins ........................................................................................... 27 1.3.3 Smad Transcriptional Activation and BMP Response Elements ........................................ 28 1.4. TGF-β and BMP Cross-talk with Major Pathways .................................................................... 29 1.4.1 BMP and the MAPK pathway ............................................................................................. 30 1.4.2 BMP mediated crosstalk with AR ....................................................................................... 31 1.4.3 BMP Crosstalk with IGF-I Signaling Pathway ................................................................... 32 3 Figures ............................................................................................................................................... 39 Chapter 2: Insulin-Like Growth Factor I Suppresses Bone Morphogenetic Protein Signaling in Prostate Cancer Cells by Activating mTOR Signaling ...........................71 ABSTRACT ...................................................................................................................................... 71 INTRODUCTION ............................................................................................................................. 72 METHODS ....................................................................................................................................... 74 RESULTS .......................................................................................................................................... 83 DISCUSSION ................................................................................................................................... 90 FIGURES .......................................................................................................................................... 93 TABLES .......................................................................................................................................... 125 Chapter 3 Smads 1 and 5 but not Smad8 are Activated by Rapamycin and Promote Cytostatic/Cell Death Responses in Prostate Cancer Cells. ........................129 ABSTRACT .................................................................................................................................... 129 INTRODUCTION ........................................................................................................................... 129 METHODS ..................................................................................................................................... 130 RESULTS ........................................................................................................................................ 137 Chapter 4 Summary, Discussion and Future Directions ..........................................183 4.1 Summary .................................................................................................................................. 183 Chapter 2: IGF-I suppresses BMP4-induced cell death through a PI3K/Akt/mTOR dependent mechanism. .................................................................................................................................. 183 Chapter 3: Rapamycin (an mTOR inhibitor) reverses mTOR-mediated inhibition of BMP signaling, thus leading to enhanced BMP-mediated activation of Smad(s) 1/5/8, Id1 transcriptional regulation, and cell death in vitro and in vivo ...................................................... 185 4.2 Discussion and Future Directions for Chapter 2 ....................................................................... 187 4.2.1 BMPRI, BMPRII, Smad1, Smad5, and Smad8-mediated interactions with mTOR, Rictor and Raptor in LNCaP cells. ......................................................................................................... 187 4.2.2 BMP4-mediated apoptosis and survivin in prostate epithelial cell lines. .......................... 188 4.2.3 IGF-I and BMP-induced apoptosis in prostate epithelial cells. ......................................... 189 4.3 Future Directions for Chapter 3 ................................................................................................. 190 4.3.1 FKBP12-mediated inhibition of BMP signaling. .............................................................. 190 4.3.2 Androgen Receptor and BMP-mediated Smad1/5/8 activation and Id1 promoter activation.191 4.3.3 Smad1, Smad5, and/or Smad8 as prognostic markers for prostate cancer. ....................... 193 4 List of Tables Table 2.1. List of BMP4 regulated genes specifically altered by IGF-I…………...127 5 List of Figures Figure 1.1. Prostate Anatomy. ...........................................................................................39 Figure 1.2. Zone of the Prostate ........................................................................................41 Figure 1.3. Anatomical staging of Prostate Cancer. ..........................................................43 Figure 1.4. Prostate Cancer Staging System. ....................................................................45 Figure 1.5. Human Prostate Cancer Progression from Normal Epithelium to Metastasis. ..........................................................................................................................47 Figure 1.6. TGF-β Superfamily Signaling. .......................................................................49 Figure 1.7. TGF-β Superfamily role in Human Cancers. .................................................51 Figure 1.8. TGF-β Superfamily Ligands ...........................................................................53 Figure 1.9. TGF-β Pathway and Human Disease. ............................................................55 Figure 1.10. BMP mediated Signaling ..............................................................................57 Figure 1.11. Dual Role of TGF-β Mediated Signaling .....................................................59