0.: . . 3%. , r 1.“.WW . . i 3.26533 Illl‘l xix??? ' .quwuuéry .. z 1.3.. I... . 3‘53.- ..) ... . {ti-R .59.?- 3...! rings. .11....Iti . a-zlutaz: $531.3... . h: 3‘ [:1- . .ll lti...l.v11. .Wfia I!!! in. in. huriifiz: 2 «1111, in ratifies. z 4 .‘ . . , .‘ .II...:{ .9», II‘ 3...... b 5 03611.1! u$.n‘ 3\ (4‘9: .‘ Err: THF‘S'S LIBRARY 3.” a Michigan State University This is to certify that the dissertation entitled ANDROGEN RECEPTOR AND INTEGRIN REGULATION OF PROSTATE EPITHELIAL DIFFERENTIATION AND TUMOR CELL SURVIVAL presented by LAURA ELAINE LAMB has been accepted towards fulfillment of the requirements for the Ph.D. degree in Cell and Molecular Biology M/CM Major Professor’s Signature SK/ 1/ /0 Date MSU is an Affirmative Action/Equal Opportunity Employer — -..-.—.-.-,-.-.-.-.-.-.-.-.—>— PLACE IN RETURN Box to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 5/08 K:/Prolecc&Pres/ClRC/DateDue.indd ANDROGEN RECEPTOR AND INTEGRIN REGULATION OF PROSTATE EPITHELIAL DIFFERENTIATION AND TUMOR CELL SURVIVAL By Laura Elaine Lamb A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Cell and Molecular Biology 2010 ABSTRACT ANDROGEN RECEPTOR AND INTEGRIN REGULATION OF PROSTATE EPITHELIAL DIFFERENTIATION AND TUMOR CELL SURVIVAL By Laura Elaine Lamb Development of strategies for more effective treatment of prostate cancer is limited by an incomplete understanding of the mechanisms regulating survival of either normal prostate or prostate cancer cells. Androgen receptor (AR) signaling plays an important role in regulating differentiation and cell survival in the prostate and in prostate cancer. Prostate cancer arises from the AR expressing epithelial cells of the prostate. Adhesion to matrix through integrins is required for survival of most epithelial cells. However, AR-expressing epithelial cells of the prostate are not adherent to matrix. Paradoxically in prostate cancer, the tumor cells both express AR and are adherent to matrix, allowing for interactions between these two signaling pathways. Our hypothesis is that the interaction of cancer cells with the matrix and the integration of signals from integrins and AR regulate their survival, while AR regulates survival of normal cells independently of integrins. We have demonstrated that PC3 and DU145 prostate tumor cell lines require PI3K signaling for cell survival. In addition, adhesion of PC3 cells to Iaminin 1 promotes survival via integrin-mediated activation of Src leading to increased expression of the pro-survival protein Bcl- xL. In DU145 cells, adhesion to collagen 1 drives survival through activation of EGFR and Erk. During prostate cancer progression, there is increased expression of the Iaminin 1-binding integrin (1601. Previous studies have demonstrated that AR can lead to increased expression of 0681, suggesting that AR may drive cell survival by altering integrin expression. We have demonstrated that expression of AR in PC3 cells can rescue cells from death induced by inhibition of PI3K when adherent to Iaminin 1. Expression of AR in PC3 cells leads to increased expression of integrin 0631 and Bcl-xL along with increased activation of NF-KB. Blocking each these components individually concurrent with inhibition of PI3K led to death of the AR-expressing cells, suggesting that AR regulates cell survival through enhancement of aSB1lNF- KB/BCI-XL signaling. AR expression in PCB tumor cells also correlates with increased filopodia formation, Src activity, and cell migration. To assess the role AR in normal cell survival, we generated an in vitro differentiation model. Confluent primary human prostate epithelial cell cultures were treated with KGF and androgen (DHT). After two weeks, a suprabasal cell layer formed in which cells no longer expressed integrins, p63, K5/14, EGFR, FGFR2lllb, or Bel-2, but instead expressed AR and androgen-induced differentiation markers, including K18/19, TMPRSSZ, ka3.1, PSMA, KLK2 and secreted PSA. Differentiated prostate cell survival depended on E-cadherin and PI3K, but not KGF, DHT, AR or MAPK. Therefore, while in the prostate tumor cell line PC3, AR and integrin 0681 cooperate to drive cell survival, neither AR nor integrins were required for survival of differentiated prostate epithelial cells. DEDICATION In loving memory of my mother, Joyce Elaine Gier, who succumbed to cancer but has continued to inspire me (1953—1995). To my husband, who supports me in all my endeavors. ACKNOWLEDGEMENTS This dissertation is the result of the support, collaboration, and encouragement from numerous colleagues, family and friends. I am extremely grateful for the guidance and mentorship of my advisor, Dr. Cindy Miranti. She has always had an open door, nurtured my scientific interests, encouraged my growth as a scientist, and has helped me achieve whatever goals I set for myself. I feel privileged to be her first graduate student. Thank you Dr. Miranti for your time, patience and knowledge- you have helped drive my passion for cancer research and to one day be a mentor myself. I know that I can count on your continued advice throughout my career. I feel incredibly lucky to have Shannon Lamb as my husband, with whom I feel everything and anything is possible. He is my rock, who supports and encourages all my endeavors, and can always make me laugh. Thank you Shannon for everything that you do and continue to do to support my dreams. I am grateful for the guidance and invaluable advice that I received from my graduate committee at MSU including Drs. Sandy Haslam, John LaPres, Bob Wiseman, and Richard Miksicek as well as investigators at VAI with whom our lab interacts including Drs. Jeff MacKeigan, Art Alberts, and Nick Duesbery as well as the members in their labs, especially Dr. Jenn White, Dr. Brendan Looyenga, and Natalie Wolters. I am also thankful for the reagents and insights of our collaborator Dr. Beatrice Knudsen (Fred Hutch Cancer Research Institute). Also, I would like to thank Dr. Melinda Frame (MSU) and Rich West (VAI) for sharing their knowledge and expertise of microscopy and flow cytommetry with rne. I work with some of the best coworkers around in the Laboratory of Integrin Signaling and Tumorigenesis including Veronique Schulz, Dr. Elly Park, Kristen Saari, Jelani Zarif, and Jessica Trahey as well as past members Dr. Mat Edick, Lia Tesfay, Dr. Suganthi Sridhar, Susan Spotts, Eric Graf, Erica Bechtel, Gary Rajah, Fraser Holleywood, Derek Janssens, and Penny Berger. I love the culture that our lab has in that we support each other, have a sense of humor, and all work hard. I am happy that I can call you all friends and will have lifelong relationships with you all. I am especially thankful to Dr. Mat Edick who spent a lot of his time training me when I first joined the lab. To my original VAI/MSU partners in crime, Charlie Miller, Aaron DeWard, and Sebla Kutluay, thank you for making this experience so enjoyable. I will miss our regular lunches together but I know that each one of you will go on to do incredible things. I know we will all look back and remember how spoiled we were here. I look forward to staying in touch and watching everyone’s career grow. To my MSU classmates, especially but not limited to Julie Bordowitz, Eric Marrotte, Katie Sian, Danielle Nevarez, Aaron McBride, Gabby Meyer, and Jamie Kopper, I appreciate all those times we spent together studying for classes in the fishbowl, going on our annual Uncle John’s Cider trip, forwarding funny journal article titles, and summer barbeques that I will treasure as part of my graduate school experience. vi I am very thankfulfor my family, including my parents who always encouraged my creativity and love for reading, nature, and science. Thank you for always supporting my dreams. To my sister Stephanie, for encouraging me to never settle. To my in-Iaws, especially my mother-in-law, for always being supportive. Lastly, I would like to thank everyone I carpooled with for getting me to and from work safely all these years. Thank you all from the bottom of my heart. ~LL~ vii TABLE OF CONTENTS LIST OF TABLES ................................................................................. x LIST OF FIGURES ................................................................................ xi LIST OF SYMBOLS OR ABBREVIATIONS ........................................... xiii CHAPTER 1 INTRODUCTION ................................................................................... 1 Introduction ................................................................................. 2 Prostate Biology and Cancer Progression .......................................... 3 Normal Biology ................................................................... 3 Prostate Cancer Progression ................................................. 6 Androgen Receptor (AR) and Prostate Cancer .................................... 8 Molecular Changes Associated with Prostate Cancer Progression ........ 14 Phosphate and Tensin Homolog (PTEN) and Pl3K/Akt Signaling.14 Nuclear factor-kappaB (N F-KB) ............................................. 17 TMPRSSZ-Erg .................................................................. 20 Integrin Expression and Signaling in Prostate and Prostate Cancer ...... 21 Integrin Signaling in Prostate and Prostate Cancer ................... 23 Limitations of Current Models ........................................................ 26 Canine and Murine Models .................................................. 26 Cell Lines ......................................................................... 30 Framework of Dissertation ............................................................ 32 References ................................................................................ 35 CHAPTER 2 lNTEGRIN-MEDIATED SIGNALING IN PROSTATE TUMOR CELLS .............. 59 Introduction ............................................................................... 60 Results ...................................................................................... 64 Discussion ................................................................................. 73 Materials and Methods ................................................................. 79 Acknowledgements ..................................................................... 84 References ................................................................................ 86 CHAPTER 3 AR-ENHANCED 0661 INTEGRIN AND BCL-XL EXPRSSION PROMOTES ANDROGEN-INDEPENDENT PROSTATE TUMOR CELL SURVIVAL INDEPENDENTLY OF PI3K SIGNALING .................................................. 92 Introduction ................................................................................ 93 Results ..................................................................................... 97 Discussion ............................................................................... 124 Materials and Methods ............................................................... 136 Acknowledgements ................................................................... 148 References ............................................................................... 149 viii CHAPTER4 E-CADHERIN—MEDIATED SURVIVAL OF ANDROGEN RECEPTOR EXPRESSING PROSTATE CELLS ........................................................ 162 Introduction .............................................................................. 163 Results .................................................................................... 165 Discussion .............................................................................. 187 Materials and Methods ................................................................ 196 Acknowledgements ................................................................... 204 References .............................................................................. 205 CHAPTER 5 CONCLUSIONS AND PERSPECTIVES .................................................. 214 Summary ................................................................................. 215 Validation of AR Effects in Additional Models .................................. 219 Other Cell Lines ............................................................... 219 In Vivo Studies ................................................................ 220 Understanding Differentiation in Prostate Epithelial Cells .................. 221 Next Generation of AR-expressing Models ..................................... 221 Therapeutic Implications ............................................................. 223 References .............................................................................. 225 Table 1. Table 2. Table 3. Table 4. Table 5. Table 6. Table 7. LIST OF TABLES siRNA Sequences ................................................................ 140 Immunoblotting Antibodies ..................................................... 142 Flow Cytometry Antibodies ..................................................... 144 qRT-PCR Primers ................................................................ 147 lmmunofluorescence Antibodies ......................................... 200 RT-PCR Primers ............................................................... 202 Summary of AR, PTEN, and Integrin 06 Expression and Survival Pathways in Prostate and Prostate Cancer Cell Lines In Vitro ............................................................................. 217 LIST OF FIGURES Images in this dissertation are presented in color. Figure 1. Integrin and AR Expression in Prostate Cancer Progression ............ 4 Figure 2. Integrin-Mediated Survival Signaling in Prostate Epithelial Cells ...... 34 Figure 3. Integrin-mediated signaling on LM1 in PC35 ............................... 65 Figure 4. PCB cells depend on PI3K and Src signaling for survival ............... 69 Figure 5. Integrin-mediated Signaling in DU145 Prostate Tumor Cells ............ 71 Figure 6. Models for PC3 and DU145 integrin-mediated survival .................. 74 Figure 7. On LMI, AR is a survival factor that acts independently of Pl3K signaling and androgen ........................................................................ 98 Figure 8. AR promotes survival through up-regulation of integrin a6 ............. 105 Figure 9. AR and integrin d6 regulate Bcl-xL expression ........................... 108 Figure 10. BcI-xL promotes survival independent of PI3K signaling ............. 110 Figure 11. AR regulates Src activity ....................................................... 112 Figure 12. AR promotes filopodia formation and migration ......................... 113 Figure 13. AR regulates integrin a6 and Bcl-xL mRNA expression ............... 115 Figure 14. AR and integrin a6 regulate NF-KB signaling ............................. 120 Figure 15. AR and integrin d6 regulate PAK1/2 signaling ........................... 125 Figure 16. Model for AR signaling in PC3 cells ......................................... 127 Figure 17. AR and AR-dependent proteins are present in the differentiated cultures... ......................................................................................... 168 Figure 18. Differentiation-specific epithelial markers present in the top cells of differentiated cultures .......................................................................... 171 xi Figure 19. Prostate epithelial differentiation is accompanied by loss of integrin expression ........................................................................................ 1 74 Figure 20. Prostate epithelial differentiation is accompanied by loss of laminin 5 expression ........................................................................................ 175 Figure 21. Differentiated cells respond to androgen .................................. 177 Figure 22. Isolation of secretory-like cells ................................................ 179 Figure 23. Signaling pathways in secretory-like cells ................................ 181 Figure 24. Secretory-like cells are dependent on PI3K and E-cadherin, but not androgen or KGF, for survival ............................................................... 183 Figure 25. AR is not required for secretory-like cell survival ........................ 185 Figure 26. Model of Differentiation ......................................................... 191 xii LIST OF SYMBOLS OR ABBREVIATIONS Abbreviation/Symbol Definition 4201 19 1 ,9-pyrazoloanthrone 3D three-dimensional A adenine or alanine (1 alpha a2 integrin a2 a3 integrin a3 a5 integrin 05 a6 integrin <16 ABI Applied Biosystems ABL Abelson murine leukemia viral oncogene homolog 1 ADT androgen deprivation therapy AF transactivation function domain AG AG1478 AG1478 Tyrphostin; N-(3-ChlorophenyI)-6,7-dimethoxy-4- quinazolinamine Akt protein kinases B AP1 activator protein 1 AR androgen receptor AR1 PC3-AR-1 AR2 PC3-AR-2 ARE androgen response element ATCC American Type Culture Collection AZ Arizona I3 beta 8 bottom layer cells b1 integrin 81 b4 integrin B4 BcI-2 B-cell leukemia/lymphoma 2 Bcl2L1 Bcl-xL Bcl-xL BCL2-Iike 1 Bclxl-1 PC3-BclxI-1 BclxI-5 PCB-Bclxl-5 Bclxl-7 PC3-BclxI-7 BCR breakpoint cluster region Bfl-1/A1 BCL2-related protein A1 Bit-1 Bcl-2 inhibitor of transcription 1 BNIP3 BCL2/adenovirus E1 B 19kDa interacting protein 3 BPE bovine pituitary extract xiii BSA Bub1 C CaCl CASP8 CCD cDNA c-FLIP ChlP ChIP-chip CK CL cm CMB COZ CRPC CSS Ct d D DAP3 DHT DMEM DMSO DNA DNAse DR Ecad Ecad Ab ECM EDTA EGF EGFR ELISA EMT ER ERE Erg ERK ETS et. al. bovine serum albumin budding uninhibited by benzimidazoles 1 homolog cytosine calcium chloride caspase 8 charge coupled device complementary DNA CASP8 and FADD-Iike apoptosis regulator chromatin immunoprecipitation chromatin immunoprecipitation with on-array detection cytokeratin collagen centimeter Cell and Molecular Biology carbon dioxide castration resistant prostate cancer charcoal stripped serum count day DMSO death-associated protein 3 dihydrotestosterone Dulbecco's Modified Eagle Medium dimethyl sulfoxide deoxyribonucleic acid deoxyribonuclease death receptor blocking E-cadherin antibody blocking E-cadherin antibody extracellular matrix ethylenediaminetetraacetic acid epidermal growth factor Epidermal growth factor receptor enzyme-linked immunosorbent assay epithelial-mesenchymal transition estrogen receptor estrogen-response element ETS-related gene extracellular signal-regulated kinase E-twenty six family of transcription factors et alii (and others) xiv ETV FACS F-actin FADD FAK FBS FGF FGFR F HC FL FN FOXO 9 G GADD45b GAPDH GTPase h HDAC HEPES HER2/ErbB HlF-1o HI-FBS HIV HO Hsp hygro IAP IF i.e. IGF IGFR IgG IkB IKK IL ILK IP IPA-3 IRB ITG ets variant Fluorescence Activated Cell Sorting filamentous polymers of actin Fas-associated via death domain focal adhesion kinase-1 fetal bovine serum fibroblast growth factor fibroblast growth factor receptor low density lipoprotein receptor full length fibronectin forkhead box-O transcription factors gamma guanine growth arrest and DNA-damage-inducible, beta glyceraldehyde 3-phosphate dehydrogenase guanosine triphosphate binding protein hour histone deacetylase 4-(2-hydroxyethyl)-1-piperazineethanesuIfonic acid human epidermal growth factor receptor 2 hypoxia-inducible factor-1d heat inactivated fetal bovine serum human immunodeficiency virus Hoechst 33258 heat shock protein hygromycin inhibitor of apoptosis immunoflourescence id est (that is) insulin growth factor Insulin-like growth factor receptor immunoglobulin G inhibitor of NF-kB lkB kinase interleukin integrin-linked kinase immunoprecipitate 1,1'-Disu|fanediyldinaphthalen-Z-ol institutional review board integrin XV ITGA6 k K K/D KGF Kip1 KLK LBD LBD18 LBDZ8 LM LN LY LY294002 M MAPK McI-1 MEM MI pL mL 1.1M mM MMP MNAR MnSOD MSP MSU mTOR Myc N Na3VO4 NaCI n.d. NaDOC NaF NEMO NF-kB ka3.1 NLS integrin a6 kappa keratin or lysine KGF and DHT treatment keratinocyte growth factor/FGF7 cyclin-dependent kinase inhibitor 1B kallikrein ligand binding domain PC3-ALBD-AR-1 8 PC3-ALBD-AR-28 Iaminin LNCaP LY294002 5-(2,2-Difluoro-benzo[1 ,3]dioon-5-ylmethylene)-thiazolidine- 2,4—dione methionine Mitogen-activated protein kinase myeloid cell leukemia seqUence 1 (BCL2-related) Minimum Essential Medium Michigan microliter milliliter micromolor millimolar matrix metalloproteinases modulator of non-genomic activity of ER superoxide dismutase 2, mitochondrial macrophage-stimulating protein Michigan State University mammalian target of rapamycin myelocytomatosis viral oncogene homolog any nucleotide or the number of experimental replicates sodium orthovanadate sodium chloride not determined sodium deoxycholate sodium fluoride NF-kB essential modulator nuclear factor kappa-Iight-chain-enhancer of activated B cells NK—3 transcription factor, locus 1 nuclear localization signal xvi NLSBO NLS4 nM NR13 NT OD P P1 P9 PAK PBS PD PD98059 PDGF PEC PhD phospho PI Pl3K PIN PIP3 PL PMSF PP PP2A PR PSA PSMA PTEN puro PVDF qRT-PCR R R1881 Rac1 Rb Rel RIPA RLU RNA RNase PC3-ANLS-AR-3O ' PC3-ANLS-AR-4 nanomolar anti-apoptotic protein NR13 not treated optical density phosphorylation or proline or PBS PD168393 PD98059 . ‘ p21 protein (Cdc42/Rac)-activated kinase phosphate buffered saline PD168393 2’-Amino-3’-methoxyflavone platelet-derived growth factor receptor prostate epithelial cell Doctor of Philosophy phosphorylation propidium iodide Phosphoinositide-3 kinase prostatic intra-epithelial neoplasia phosphatidylinositol (3,4,5)-trisphosphate PC3-pLKO phenylmethylsulphonyl fluoride PC3-puro serine/threonine protein phosphatase 2A progesterone receptor prostate specific antigen prostate specific membrane antigen Phosphate and Tensin Homolog puromycin polyvinylidene difluoride membrane quantitative reverse transcriptase polymerase chain reaction adenine or guanine methyltrienolone Res-related C3 botulinum toxin substrate 1 retinoblastoma 1 v-rel reticuloendotheliosis viral oncogene homolog Radio lmmunoprecipitation Assay Relative Luminescence Units ’ ribonucleic acid ribonuclease xvii RPMI rRNA RTK RT-PCR RU SB 83202190 SCT SDS SDS-PAGE Ser SF M shRNA siA6 siAR siRel siRNA siSrc si-xL SNP SP Sp1 Spi2A Src SRD5A2 starv. STR SU SU6656 SV40 SYBR green TBS TBST TCL TGF Thr Roswell Park Memorial Institute Ribosomal RNA receptor tyrosine kinase reverse transcriptase polymerase chain reaction RU486 SB202190 4-[4-(4-FluorophenyI)-5-(4-pyridinyl)-1 H-imidazoI-Z-yl]phenol scram Sodium dodecyl sulfate sodium dodecyl (Iauryl) sulfate-polyacrylamide gel electrophoresis senne serum free media short hairpin RNA siRNA against A6 siRNA against AR siRNA against p65 (ReIA) small interfering RNA siRNA against Src siRNA against BcI-xL single-nucleotide polymorphism serine protease Sp trans-acting transcription factor 1 serine (or cysteine) peptidase inhibitor, clade A, member 36 sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog steroid 5-alpha reductase starvation staurosporin SU6656 2,3-Dihyd ro-N,N-dimethyl-2-oxo-3-[(4,5,6,7-tetrahydro-1 H- indoI-2-yl)methylene]-1 H-indole-5-sulfonamide Simian virus 40 N',N'-dimethyl-N-[4-[(E)-(3-methyl-1 ,3-benzothiazoI-2- ylidene)methyI]-1-phenquuinolin-1-ium-2-yI]-N-propylpropane- 1,3-diamine large T antigen, thymine, or top layer cells small t antigen Tris-buffered saline Tris-buffered saline containing 0.1% Tween 20 total cell lysate transforming growth factor Transmembrane protease, serine 2 xviii TMP TMPRSSZ TN TNF TRAMP Tub TUNEL U U0 U0126 uPAR UV VAI VARI veh W WA WCL X XIAP Y zVAD TMPRSSZ transmembrane protease, serine 2 Tennessee tumor necrosis factor transgenic adenocarcinoma mouse prostate tubufin Terminal Deoxynucleotide Transferase dUTP Nick End Labefing units U0126 1,4-Diamino-2,3-dicyano-1,4-bis(o- aminophenylmercapto)butadiene monoethanolate plasminogen activator, urokinase receptor ultraviolet Van Andel Institute Van Andel Research Institute vehicle adenine or thymine Washingtom whole cell lysate any amino acid X-Iinked inhibitor of apoptosis cytosine or thymine , or tyrosine carbobenzoxy-vaIyl-alanyI-aspartyl-[O-methyl]- fluoromethylketone xix CHAPTER ONE: INTRODUCTION Introduction Prostate cancer is the most diagnosed malignancy and second leading cause of cancer related death in American men. In 2009, it is predicted that in the United States alone there will be 192,280 new cases (approximately 1 in 6 men) and 27,360 prostate cancer-related deaths (approximately 1 in 35 men) (1, 2). Prostate cancer incidence strongly correlates with age and usually is detected in men sixty or older (3, 4). Autopsy studies suggest that up to 80% of 80 year old men have prostate cancer and some experts predict that all men would develop prostate cancer if they lived long enough (3, 4). However, prostate cancer is a relatively slow growing tumor and the majority of men diagnosed with prostate cancer will actually die from other causes (5). While primary prostate cancer is highly treatable by surgical resection and radiation, prostate cancer that has metastasized is not. This is reflected by a five-year survival rate of 100% for local and regional prostate cancer, and 31% for metastatic prostate cancer (6). Androgen signaling is critical in prostate cancer development and disease; androgen deprivation therapy (ADT) is the primary therapy for metastatic prostate cancer (7). Also known as androgen ablation or androgen suppressive therapy, ADT reduces levels of circulating androgen in the body in combination with blocking ligand binding to AR. While this is initially clinically successful and results in rapid tumor regression, it is not curative. Ultimately the prostate cancer will recur and will no longer respond to ADT. This is known as castration-resistant prostate cancer (CRPC), hormone-refractory, or androgen-independent prostate cancer. Currently, there are no curative therapies for CRPC and patients ultimately succumb to the disease. Prostate Biology and Cancer Progression Normal Biology The human prostate is an encapsulate glad about the size of a walnut located inferior to the bladder (5). It surrounds the urethra and functions to produce secretory alkaline proteins for the seminal fluid which nourish and protect the sperm (5). Although the prostate is thought to share common embryonic origins as well as the same blood supply as adjacent male urogenital tissues, these surrounding tissues develop ne0plasia only in rare cases (5). The prostate has a ductal-acinar histology, with stromal cells throughout and epithelial cells surrounding the acini. The epithelium of the human prostate consists of two cell layers, a basal layer and a secretory layer. The prostate basal layer is mostly made of basal epithelial cells as well as a small subpopulation of neuroendocrine cells. The basal cells are mitotic and adhere to a basement membrane containing collagens IV and VII, and laminins 5 and 10/11 (8-10) (Figure 1). Prostate basal cells give rise to terminally differentiated secretory cells (8-10). The secretory cells, also known as luminal cells, are located suprabasally and face the lumen of the prostate (Figure 1). There is also a stromal compartment of the prostate which contains myoepithelial cells, FIGURE 1. AR, integrin, and matrix progression in prostate cancer progression. (Top panel) The prostate consists of a stromal and epithelial compartment separated by matrix composed of LM5, LM10, CLlV, and CLVll. The basal epithelial cells are adherent to this matrix by integrins 0684, 0381, and 0281. The secretory epithelial cells express integrin 0681 but are not adherent to matrix. They express AR and secrete proteins into the lumens of the prostate. (Middle panel) In primary prostate cancer, the cells are adherent to LM10 and CLlV matrix via integrin 0681 and express AR. (Bottom panel) In metastatic prostate cancer, AR is often overexpressed or mutated. Cells are adherent to new, different matrices such as CLl, LM1, and FN as they spread to new sites in the body, primarily bone. Cell lines derived from metastatic lesions express integrins 0281, 0381, 0581 and 0681. FIGURE 1 Normal Gland lumen E Secretory AR+ 0681 8 ”3* Basal AR- 0684, (1381, 0281 ECM LM5, LM10, CLIV, AR+ CLVII Stromal Primary Prostate Cancer 0681 LM10, CLIV Metastatic Prostate Cancer AR++ mutations ((XZBI, G3B1, (l5B1, (1.631) CLI, LM1, FN fibroblasts, and blood vessels (Figure 1). Unlike other epithelia, prostate epithelial cell differentiation is regulated by androgen signaling (11-16). AR is expressed only in the differentiated secretory cells and not in the basal cells, however AR is also expressed in the stroma (17) (Figure 1). Elegant tissue recombination studies by Cunha and colleagues demonstrated that AR is required in the mesenchyme, but not epithelium, for prostate duct development (18-20). However, androgen signaling in the epithelium is required for prostate function (18-20). Androgen signaling in the secretory cells drives AR-mediated transcription of proteins, such as prostate specific antigen (PSA/KLK3), which are secreted into the lumen of the prostate. In the normal prostate, adhesion to matrix and expression of AR are mutually exclusive. In addition to AR expression and adhesion to matrix, differentiation of basal cells into secretory cells also correlates with changes in cytokeratin, growth factor receptor, and integrin expression (discussed in more detail in Chapter Four). Prostate Cancer Progression Prostate cancer arises from the epithelial compartment and appears to be associated with a dysplastic lesion referred to as prostatic intra-epithelial neoplasia (PIN) (5). In low grade PIN, abnormal AR positive cells are observed in the secretory layer (21). However, as PIN progresses, there appears to be a loss of basal cells allowing the AR positive carcinoma cells to adhere to matrix (21). The loss of the basal cells is hypothesized to be due to invasion or over- proliferation of the carcinoma cells into the basal cell layer, or due to death of the basal cells themselves (22, 23). High grade PIN precedes the appearance of prostate cancer usually by five to ten years (21). These lesions are heterogenic and multifocal; PIN can be immediately adjacent to and within the same acini structure as normal epithelium and PIN fails to permeate into the stroma (21). Although the cell of origin for prostate cancer remains controversial, some lines of evidence strongly support that it is from a secretory or secretory precursor cell. For one, all primary prostate cancer is positive for AR, suggesting it must arise from AR positive cells, and not negative basal cells. Furthermore, secretory cells first appear during puberty when there is a dramatic and permanent increase in circulating testosterone; there are no reported cases of prostate cancer occurring before puberty and the appearance of secretory cells (4, 21). Unlike many other solid tumors, the vast majority of primary prostate cancer grow slowly and are asymptomatic, and may take years to develop. Hence some elderly patients with other health considerations may choose “watchful waiting” rather than surgery or radiation. Prostate cancer metastasizes primarily to the bone (usually the spine and ribs) and is often the only clinically detectable site of metastasis (24). Unlike most other cancers that metastasize to bone, prostate cancer is osteoblastic (bone forming) rather than osteolytic (bone lysing), which is often painful for the patient (24). Prostate cancer also commonly metastasizes to the lymph nodes, brain, and lungs, although it can spread to other sites as well. Androgen Receptor (AR) and Prostate Cancer AR is critical for prostate cancer viability and growth (7, 25). Inhibiting AR expression within animal models of prostate cancer inhibits tumor development, progression, and leads to recession of established tumors (26-28). Studies in vitro demonstrate that inhibition or loss of AR using antibodies, ribozymes, or siRNA can inhibit prostate cancer proliferation and survival (26, 29—32). Thus AR is a potent mediator of cell survival and growth in prostate cancer. AR is a nuclear transcription factor activated in response to the steroid hormone androgen (17). It consists of an N-terrninal transactivation domain (AF- 1) that can function in the absence of ligand, a C-terminal ligand binding domain (LBD) associated with a second transcriptional regulatory function (AF-2), and a DNA binding domain between the two. Classically, non-ligand bound AR is localized in the cytoplasm bound to molecular chaperones including Hsp90 and Hsp70, which confonnationally prevent the nuclear localization signal (NLS) on AR from being exposed. This also keeps AR in a high-affinity state for ligand binding (33). Circulating androgen (testosterone) enters the prostate cells and is converted to dihydrotestosterone (DHT) by the enzyme 50-reductase (SRD5A2). DHT has a 5X higher affinity for AR compared to testosterone. This induces a conformational change In AR, allowing disassociation from Hsp70/90, homodimerization, exposure of the NLS, and subsequent translocation into the nucleus. Ligand binding of AR does not happen in cell Iysates, suggesting that this is not protein autonomous, but rather requires additional cellular factors (34). Once in nucleus, AR binds DNA at specific sequences referred to as androgen response elements (AREs) to regulate gene expression. AREs are typically two 6 base pair “half-sites” that are direct or inverted repeats separated by 3 base pairs (i.e. GGTACAnnnTGTTCT) (35). These sites are highly degenerate and divergent, making ARE prediction in gene promoters difficult (36). Microarray and chromatin immunoprecipitation (ChlP) with on-array detection (ChlP-chip) experiments suggest that as many as 20-30% of AR regulated genes do not contain predicted ARE sites (37-41). Once bound to DNA, AR can bind co-activators, co-repressors, and other transcriptional modifiers to either activate or repress transcription of target proteins (reviewed in 42, 43, 44). It has been suggested that AR is a more gradual activator of transcription after ligand stimulation compared to other steroid receptors like estrogen receptor (ER), consistent with long range interactions between enhancer elements and promoter regions (45-47). However, this has only been investigated in the regulation of a few genes and does not necessarily exclude a role of AR from having more immediate effects. Known AR targets include prostate-specific antigen (PSA/KLK3), KLK2, and TMPRSSZ (48, 49). These are often increased during prostate cancer progression; PSA detection is currently used as a marker in prostate cancer screening and for monitoring the effectiveness of ADT. However, limitations of PSA testing such as lack of specificity, elevation in benign disease, failure to detect PSA-negative tumors, and inability to distinguish slow growing tumors from aggressive ones, makes prostate cancer biomarker discovery an important area of research (50). Furthermore, during prostate cancer progression, there is a switch in AR function. In normal prostate cells, AR functions to drive differentiation and expression of the secretory proteins of the prostate, as well as suppress cell growth (16, 51-53). In prostate cancer, AR drives proliferation and cell survival (7, 25-32). The mechanism driving this conversion is unknown but likely involves cooperation of AR with other oncogenic events. All existing ADTs are designed to prevent AR signaling by blocking androgen binding to AR. This is done through two mechanisms. The first is to reduce systemic androgen levels in the body using chemical castration agents such as gonadotropin-releasing hormone agonists (i.e. leuprolide). The second mechanism is direct competition using competitive antagonists for AR binding (i.e. bicalutamide, flutamide). Limitations of these current generation antagonists include a 30-fold weaker affinity for AR than DHT (54) and that they do not prevent AR localization to the nucleus (55). Both of these strategies are used in combination during ADT. ADT causes remission in 80-90% of patients undergoing therapy, resulting in a median progression-free survival of 12 to 33 months (56). After remission the recurrent cancer is castration-resistant and the median survival is 23—37 months from the time of initiation of ADT (56). Currently, the next generation of AR antagonists, which are non-competitive or have a greater AR affinity, are being developed (34, 57). AR can be post-translationally modified by phosphorylation, acetylation, sumoylation, and ubiquitination (58-66). Post-translational modification of AR can affect its stability, ligand binding affinity, protein interactions, or 10 transcriptional activity. These have been mostly described in vitro and have not necessarily been observed in vivo. However, in vivo detection can be difficult and transient modifications or low levels of modification may not necessarily be detected. Furthermore, mutations of some of these modification sites fail to alter AR's activity, stability, or sensitivity to ligand as expected. One of the few phosphorylation sites that has been reproducibly observed, in both in vitro and in vivo, to have an effect is Ser650, in which phosphorylation stimulates nuclear export (58, 67). Androgen-induced phosphorylation of Ser308 has also been reproducibly demonstrated to increase the transcriptional activity of AR (68). In CRPC, AR promotes survival and proliferation, even in the absence of physiological levels of androgen. Several mechanisms have been proposed to explain this phenomenon (reviewed in 7). Other steroid receptors, including ER and progesterone receptor (PR), have been reported to be involved in non- genomic signaling, which can also have important consequences on cell behavior (69, 70). These non-genomic mechanisms are associated with rapid responses (within seconds) to ligand stimulation. This has been less well characterized for AR, but non-genomic signaling may be a mechanism by which AR can function in CRPC. AR has been reported to be in a complex with Src and ERO via its PXXP domain (71). This can lead to rapid androgen-induced mitogen-activated protein kinase (MAPK) activation and has been reported to regulate cell survival and proliferation, although the mechanism of this remains unclear (71). However, this interaction does require androgen and does not explain disease progression in CRPR. AR can also form a complex with Src and 11 MNAR (modulator of non-genomic activity of ER) (72). This complex is androgen dependent in LNCaP cells but is constitutively active in a castration-resistant LNCaP derivative subline. This complex activates Src and MAPK/ extracellular signal-regulated kinase (ERK) signaling (72). Hence, this may contribute to the development of CRPC. During ADT, physiological levels of androgen are greatly reduced, but low levels of androgen (in the nanomolar range) can still be detected in tissues (73, 74). Overexpression of AR sensitizes castration-resistant prostate cancer cells to the low levels of androgens (75). Furthermore, AR is amplified and overexpressed in 20-30% of CRPC (39, 55, 76, 77). Hence, the low levels of androgens present in tissues may be sufficient for AR function. It is speculated that the source of this is the adrenal glands, which can produce testosterone and are not influenced by current ADTs. However, mice do not produce adrenal androgens, so castration results in “complete androgen ablation” (78). Furthermore, mice can still develop CRPC and AR is still found in the nucleus of these cells. This suggests that residual androgen production after castration is not playing a crucial role in driving AR function in CRPC (67, 79, 80). Alternatively, it has been proposed that prostate cancer cells can synthesize their own androgen from cholesterol (81). Currently, this has only been demonstrated in vitro and it remains to be determined if this occurs in vivo. Another mechanism is via gain-in-function mutations in AR which allow other steroids, such as corticosteroids and anti-androgens, to bind and activate AR; similar to what has been seen in breast cancer. Mutations in AR are 12 generally not seen in prostate cancer until after ADT failure; AR mutations are observed in approximately 10-25% of CRPC (82, 83). Alterations in AR co- regulators can also contribute to CRPC in that these changes have been demonstrated to drive AR nuclear translocation and DNA binding (55, 84-88). Crosstalk with growth factor pathways can also lead to activation of AR in the absence of ligand (25). For example, insulin-like growth factor (IGF) signaling can lead to dephosphorylation of AR at Ser650, allowing AR to remain in the nucleus (67). It can also stimulate AR transcription; stimulation with IGF-1 can induce a 5X increased in PSA secretion in LNCaP cells (89). AR crosstalk with Pl3K/AKT, NF-KB, and Src will be elaborated on below. Lastly, John lsaacs and others have suggested that CRPC develops from a subpopulation of cancer cells that never did express AR, and hence these “cancer stem cells” are not affected by ADT (90, 91). Rather the progeny of these cells undergo partial differentiation to express AR and comprise the majority of the tumor. During ADT, the AR- negative cancer stem cells have a growth and survival advantage compared to the AR-positive cancer cells, allowing the prostate cancer to reoccur after ADT. In summary, in normal prostate cells, AR function is androgen dependent and drives differentiation, secretory protein expression, and suppresses cell growth. AR signaling is critical in prostate cancer progression where it promotes cancer growth and survival. While ADT is initially successful, patients will fail and the cancer will reoccur. AR signaling is still important despite low physiological levels of androgens. This may be due to hypersensitivity to low amounts of androgen or other non-androgen ligands, amplification, overexpression, or gain- 13 r in-function mutations of AR or its co-regulators, and crosstalk with other pathways that are also misregulated in cancer. Molecular Changes Associated with Prostate Cancer Progression Prostate cancer is heterogeneous and multifocal (5, 92). The EGFR mutations in lung cancer, HER2/ERBBZ amplification in breast cancer, and BCR- ABL gene fusion in chronic myelogenous leukemia have all been demonstrated to be important in progression of a subset of these diseases and have allowed for the development of targeted therapies; however there is not a single causative mutation or common molecular change yet identified in prostate cancer (92). Even different foci within the same patient often have different genetic alterations, suggesting that prostate cancer can develop through multiple mechanisms (5, 93). This variance may be one explanation as to why the majority of prostate cancers are slow growing and will have a low likelihood of progression while a subset will progress to an aggressive disease resulting in a slow, painful death. However, some molecular studies have identified some candidate genes that may be involved in prostate cancer development and metastasis. It is important to note that few prostate cancers have all of the molecular changes included below. Phosphate and Tensin Homolog (PTEN) and PI3K/Akt Signaling Phosphate and tensin homolog (PTEN) is a phosphatase that dephosphorylates phosphatidylinositol (3,4,5)-trisphosphate (or PIP3) which 14 antagonizes of phosphatidylinositol 3-kinase (PI3K) signaling. Pl3K signaling through the downstream PIP3 target Akt (also referred to as protein kinase B) can promote cell survival, proliferation, adhesion and spreading, and thus up- regulation of PI3K/Akt signaling can provide numerous growth advantages to a cancer cell (94). PI3K activation of Akt can promote cell survival via multiple mechanisms including phosphorylation and inhibition of the pro-death proteins Bad, Bax, forkhead box-O transcription factors (FOXO), death-associated protein 3 (DAP3), and pro-caspase 9, through increased expression of the pro-survival protein survivin, and by regulating NF-KB and mTOR signaling (95, 96). Hence, PTEN acts as a tumor suppressor and inactivating mutations or loss of heterozygousity are common in many cancers including glioblastoma, breast, ovarian, colon, and endometrial cancer (97). PTEN is lost in ~30% of clinical prostate cancers and in ~60% of metastatic cancers, resulting in constitutive activation of PI3K signaling (98-100). While Akt is not amplified or overexpressed, Akt kinase activity is increased in primary prostate cancer. It is significantly increased in poorly differentiated primary prostate tumors compared to well- and moderately- differentiated tumors, is associated with advanced prostate cancer development, and is predictive of ADT failure and prostate cancer reoccurrence (101-103). Pten-deficient mice develop high-grade PIN, but without progression to invasive cancer (104). Therefore, PTEN loss is not sufficient for prostate cancer development. Overexpression of Akt and AR, but not Akt or AR alone, is sufficient to initiate and drive castration-resistant prostate 15 cancer progression in mice (105). Thus, these observations suggest a strong connection between AR and the PI3K/Akt pathway in prostate tumorigenesis. The PI3K/Akt pathway has been reported to interact with AR by both direct and indirect mechanisms (reviewed in 106). Akt regulates AR transcription (105, 107-109), stability and expression (110, 111), and post-translational modifications (112, 113). In turn, PI3K/Akt can also be activated by AR through genomic and non-genomic mechanisms (106, 114). Many prostate cancer cell lines are dependent on PI3K/Akt signaling for survival, and in some contexts this can be rescued by AR signaling. While the androgen sensitive prostate cancer cell line LNCaP undergoes cell death within 24 hours of inhibiting Pl3K/Akt pharrnacologically, addition of androgen can rescue survival (115). Long term androgen ablation of LNCaPs results in cells resistant to PI3K/Akt inhibition, and androgen-independent cell lines are less sensitive to Pl3K/Akt inhibition (116). Often, Akt activity is significantly increased in androgen-independent derivates of LNCaP cells (117, 118). Also, in vivo prostate regeneration studies demonstrate that AR and Akt signaling can synergize to promote tumor formation even after androgen ablation (105). Thus AR signaling, in some contexts independent of androgen stimulation, may promote survival independent of PI3K signaling by either regulating the same downstream targets of the PI3K pathway, or through a novel mechanism. AR can also phosphorylate and inhibit FOXO proteins, thus promoting survival independent of PI3K by regulation of a common downstream target (119). The pro-survival protein Bcl-xL can also promote survival independent of Pl3K 16 signaling in prostate cancer cells; however the mechanism by which Bcl-xL is regulated remains undetermined (120). In summary, AR along with PI3K/Akt signaling may cooperate during the progression of prostate cancer, and in some contexts, in CRPC. Nuclear factor-kappaB (NF-KB) Nuclear factor-kappaB (NF-KB) is a family of transcription factors (p50/p105, p52/p100, RelA, c-Rel, RelB) that classically are key regulators of pro-immune and inflammatory gene expression, but have also been demonstrated to be important regulators of cell growth, differentiation and survival (121-123). The prototypical heterodimer consists of p65 (RelA) and p50, but heterodimers and homodimers of p52, c-Rel, and RelB also exist and this diversity allows for distinct DNA-binding specificity and protein-protein interactions at promoter sites, allowing specific gene activation profiles under different physiological conditions. There is some overlapping gene regulation, but in general NF-KB dimers bind DNA sites with the general motif 5’- GGGRNWYYCC-3’ (where R= A or G; N= any nucleotide; W= A or T; Y= C or T) (121). NF-KB is held inactive in the cytoplasm bound to inhibitors of NF-xB (les). Stress stimuli or other environmental clues leads to formation of the IKB kinase (IKK) complex, which consists of the catalytic kinase subunits IKKO and/or IKK8 along with two molecules of the scaffold protein NF-KB essential modulator (NEMO). The IKK complex then phosphorylates IKB at two serine residues, 17 leading to its ubiquitination and subsequent proteosomal degradation. The NF- KB dimers are then free to enter the nucleus and drive transcription of target genes(121) NF-KB signaling is classically associated with driving cell survival. Mice deficient in RelA, both IKKO and IKK8, or NEMO are embryonic lethal, probably due to extensive apoptosis of hepatocytes (124-128). Overexpression of the potent pro-survival and NF-xB-regulated protein Bcl-2 is unable to rescue liver degeneration in ReIA-knockout mice, indicating that the pro-survival activity of RelA is through multiple mechanisms (129). Indeed, NF-KB signaling drives transcription of numerous pro-survival genes targeting both intrinsic and extrinsic death pathways including Bcl-2, Bcl-xL, Bfl-1/A1, NR13, c-IAP, XIAP, survivin, c- FLIP, GADD458, and Spi2A as well as the antioxidant proteins MnSOD and FHC (reviewed in 130). NF-KB signaling can also repress transcription of the pro- death proteins BNIP3, caspase 8, DR4 (TRAIL-R1) and DR5 (TRAIL-R2) (130). However it is important to note that under certain conditions, such as radiation or treatment with chemotherapeutic drugs, NF-KB signaling can drive cell death through repression of pro-survival genes including Bcl-xL and driving expression of death receptors and their ligands by altering the NF-KB heterodimer composition, recruiting the transcriptional repressor HDAC to transcription sites, or altering NF-xB localization along with other mechanisms (130). NF-xB signaling is important in the development, progression, and chemoresistance of many cancers, including prostate cancer (reviewed in 131). Increased NF-xB activity is associated with prostate cancer progression (132), 18 castration-resistance (133, 134), poor prognosis (135, 136), biochemical failure (i.e. PSA relapse) (137, 138), and has been determined to be significantly misregulated in metastatic prostate cancer based on microarray studies (139). NF-xB can regulate AR by co-stimulating AR transcription (140-142) (133) or by regulating AR expression (140, 143, 144). In androgen-dependent cell lines, NF-KB signaling can inhibit proliferation (144) and there is generally a low level of basal NF-xB activity (145). However, in androgen-independent cell lines, NF-KB signaling is often constitutively active (145), possibly due to constitutive activation of IKK0 (146). Constitutive activation of NF-xB in vivo is sufficient to maintain prostate cancer cell growth after castration in mouse models, presumably by maintaining high levels of nuclear AR (134). This suggests that there may be a functional change in NF-KB signaling in the transition to CRPC that promotes AR function independent of ligand. PI3K/Akt signaling crosstalk with NF-KB signaling has also been described. PI3K/Akt can phosphorylate and activate lKKs, turning on NF-KB signaling (147, 148). NF-KB signaling in turn can lead to increased AR transcription independent of ligand; this has been implicated in the development of CRPC in mice models (149). Cooperative crosstalk between AR, PI3K/Akt, and NF-KB has also been described in prostate cancer cells in the regulation of interleukin-4 (IL-4) expression (150). In summary, NF-KB signaling has strong correlations in the clinic with prostate cancer progression, can be activated by PI3K/Akt signaling, and can 19 stimulate AR function during castration, suggesting it plays a critical role in this disease. TMPRSSZ—Erg TMPRSSZ is a direct transcriptional target of AR. In prostate cancer, approximately 40-60% of tumors have a TMPRSSZ-Erg rearrangement, which allows for AR regulation of the ETS-related gene (Erg) and subsequent aberrant expression in prostate cancer cells (151, 152). Erg proteins are transcriptional factors that regulate cell proliferation, differentiation and tumorigenesis (153). Additionally, ETS family members ETV1, E TV4 and ETV5 have also been shown to be genetically rearranged in prostate cancer. While the biological consequence of Erg overexpression in prostate cancer has not yet been determined, it has been shown to lead to activation of the oncoprotein Myc and to repress the expression of some prostate differentiation makers (i.e. PSA, SLC45A3 (Prostein), MSMB) (151). Furthermore, there appears to be a strong correlation between TMPRSSZ-Erg expression and PTEN loss. Most ERG- positive samples have reduced or absent PTEN expression (154, 155). Transgenic mice expressing Erg fail to deveIOp PIN or develop tumors prostate tumors. However, crossing these mice with mice that were either Pten-deficient or had prostate-specific high Akt activity, resulted in offspring that developed PIN, and in the mice with a B6 background but not FVB, progression to prostate cancer was a consistent feature in offspring (154, 155). Hence, TMPRSS2-Erg 20 may be important in prostate cancer by allowing androgen regulation of the transcription factor Erg. Integrin Expression and Signaling in Prostate and Prostate Cancer Integrins are a large family of transmembrane, cell-surface receptors that bind components of the extracellular matrix (ECM) including collagen (CL), Iaminin (LM), fibronectin (FN), etc. (156). Upon ECM binding integrins mediate cellular adhesion, spreading, migration, proliferation, and survival by reorganizing the cytoskeleton and activating intracellular signaling pathways (157). Integrins exist as heterodimers comprising one alpha subunit and one beta subunit, the combination of which determines the ECM protein the cell can associate with. Integrins are required for survival in many cell types, and interruption of integrin binding to matrix results in a form of cell death termed anoikis (158). Integrins have no intrinsic catalytic activity, but rather interact with other structural and signaling proteins including kinases such as integrin-linked kinase (ILK) and focal adhesion kinase (FAK) via their cytoplasmic tall (159). Furthermore, integrins can activate receptor tyrosine kinases (RTKs) in the absence of exogenous ligand (160-167). Some examples of RTKs activated by integrins include epidermal growth factor (EGFR), platelet-derived growth factor receptor (PDGF R), the macrophage-stimulating protein receptor RON, and the hepatocyte growth factor receptor, c-Met (163, 168-171). 21 Integrin cross-talk with other signaling molecules allows coordinated cellular responses to extracellular signals such as matrix attachment and growth factors (172). Our work and that of others have demonstrated that integrin engagement is sufficient to activate receptor tyrosine kinases (160-167). For example, the macrophage-stimulating protein (MSP) receptor RON can be activated by 81 activation of c-Src independent of its ligand MSP (163). In addition, MSP enhances binding to collagen, suggesting cooperation and synergy between these two pathways (163). In another example, signaling from both integrin-mediated activation of EGFR and ligand activation of EGFR is required for cells to proliferate (162, 169). Integrin engagement of fibronectin (FN) in epithelial cells results in phosphorylation of a different subset of tyrosine residues on EGFR than ligand binding does and is enough to initiate G1 entry and progression to mid-G1 in the absence of growth factors (162). S phase entry requires EGF or serum, though over-expression of EGFR allows cell cycle progression in the absence of ligand, which has interesting implications in cancer where RTKs are often over-expressed (162, 169). Integrins can activate the PI3K/Akt pathway to promote cell survival (173, 174). Conversely, increased Akt activity can prevent anoikis and promote cell migration (95, 175). Integrin 0684 adhesion to LM5 can lead to increased activation NFKB, MAPK, and Jnk (176, 177); targeted deletion of the 84 cytoplasmic tail in keratinocytes of mice inhibits their growth and migration (177). Therefore, cross-talk has important biological effects in coordinating cellular response from multiple signaling pathways and allowing different cellular responses depending on the context. 22 While not extensively studied, steroids have also been implicated in participating in cross-talk with integrins. Integrin 0281- or 0681-mediated adhesion to CLIV or LM1 respectively has been shown to regulate ERO expression and its subsequent function in mouse mammary epithelial cells, but adhesion does not regulate ERO expression in mammary fibroblasts (178). ER may also regulate 02 integrin since the gene promoter for 02 contains estrogen- response elements (EREs), which would suggest a positive feed-back loop of regulation; however this remains under investigation (178, 179). Recently, estrogen has been shown to enhance integrin 05 expression through ERO-Sp1 interaction (180). Also in MDA-MB-231 breast cancer cells expressing PR, progesterone treatment resulted in cell spreading, increased cell attachment, and increased phosphorylation of FAK and paxillin (181). Blocking 81 with antibody caused reduced spreading and significantly inhibited adhesion to ECM, suggesting that 81 plays an important role in progesterone-induced focal adhesion (181). Integrin expression and signaling is aberrant in many cancers. For example, overexpression of LM1 can increase colon tumor growth in mouse models (182). Integrin 81 is aberrantly expressed in breast cancer and has been shown to play a central role in growth, apoptosis, invasion, and metastasis. Blocking integrin 81 with antibody has been shown to either reverse the malignant phenotype of breast cancer cells (183), or to inhibit proliferation and induce apoptosis in 3D and xenograft mice models (184). Since animals did not 23 appear to suffer any side effects, this suggests that blocking integrins may be a therapeutic option in patients (184). lntegn'n Signaling in Prostate and Prostate Cancer Prostate cancer arises from the prostate epithelium which comprises basal and secretory layers. The basal layer uses integrins 0281, 0381, and 0684 for adhesion to a basement membrane consisting of LM5 and LM10 as well as CLIV and CLVII (185). This binding of integrins to the ECM regulates survival in the basal cells (186, 187). The secretory cells do not contact a basement membrane, but rest on top of the basal cells from which they differentiate. The secretory cells only express integrin 0681; however they do not use this integrin to adhere to matrix (188). Rather, 0681 is localized to cell-cell junctions and its function there is currently unknown. In prostate cancer, there is a loss of the basal epithelial cells and prostate cancer cells are adherent to a basement membrane containing LM10 and CLIV; LM5 and CLVII are absent (185). Normal basal epithelial cells secrete and organize a LM5 matrix, so loss of these cells contributes to the altered ECM in prostate cancer. Furthermore, 0681 is the primary integrin expressed in both primary prostate cancer and lymph node metastases (189-195). Prostate cancer cell lines that form invasive tumors in mice have increased integrin 0681 expression; blocking integrin antibodies can inhibit their invasion in vitro and in vivo (196-199). One of the 22 SNPs currently associated with prostate cancer is 24 within the first intron of integrin 06 (200). This SNP lies within the epithelial- mesenchymal transition (EMT)-specific splice site in integrin 06; thus the integrin 06 in epithelial cells becomes altered when the cells undergo EMT and mesenchymal specific splice factors are being activated (200). In addition, a truncated form of integrin 06 (06p) is increased during prostate cancer progression by cleavage and removal of the extracellular domain by the extracellular urokinase plasminogen activator receptor (uPAR) (201, 202). This is thought to make the cell more motile since the integrin can no longer interact with matrix, and may contribute to cancer invasion and metastasis (203). This is supported from experiments where blocking 06 cleavage with antibody prevents bone metastasis in a mouse model (188). Furthermore, two variants of integrin 81, 81C and 81A, have been shown to be expressed in prostatic epithelium. Interestingly, integrin 81C expression is decreased in prostate cancer (204, 205), whereas integrin 81 A is increased in prostate cancer and in a mouse model for prostate cancer progression (206, 207). Integrin 81A stimulates proliferation in vitro (207). Integrin 06p81A may also alter integrin-mediated signaling with other molecules, contributing to tumorigenicity. Interestingly, ADT results in increased 81c mRNA, suggesting a possibility of androgen regulation (208). During metastasis, additional changes in integrin and ECM expression correspond to the cells’ ability to survive in distinct environments. LNCaP that were selected for increased integrin 0281 and CLl adhesion develop tumors when injected into 25 tibia of nude mice, whereas parental LNCaP cells do not (209). Integrin 0v83 or 0v85 can drive survival, migration, and metastasis in prostate cancer cell lines and xenograft models (210-213); however these integrins have not necessarily been reported in vivo and may be an artifact from culturing cells in serum. Expression of AR in PC3 cells has been shown to decrease 0684, 0381, and 0281 expression along with a reduction in adhesion to collagen and Iaminin (214, 215). Expression of AR in DU145 cells decreased 0684, but increased expression of 0281 (216). Lastly, DHT stimulation of AR expressing immortalized non-tumorigenic rat prostate CA25 cells leads to increased integrin 06 and p21-activated kinase (PAK) activity (15). This suggests that AR can regulate integrin expression. Since integrin-mediated adhesion can regulate growth factor signaling pathways including PI3K/Akt, MAPK, and NF-xB signaling, AR and integrins can potentially crosstalk through joint regulation of these pathways. These mechanisms of cross-talk between integrins and AR are largely unexplored. Limitations of Current Models Canine and Murine Models A significant challenge in understanding prostate cancer development and biology is the limitations of the models in which to study the disease. Besides humans, dogs are the only mammal in which spontaneous development of prostate cancer has been well documented (5, 217, 218). Nobel laureate 26 Charles Huggins used canines as a model of prostate cancer early in his career. Canine prostate cancer has many clinical and histological features in common with human prostate cancer including metastasis to bone, however there are some important differences (217). Canine prostate cancer is rare but aggressive, whereas human prostate cancer is usually slow growing (217). Most significant is that canine prostate cancer has little to no dependence on androgen and many canine prostate cancers do not even express AR (217, 219). In fact, castration is associated with increased prostate cancer risk in dogs although it can be protective if done early in life (217, 220, 221). These differences, along with the long lifespan, heterogenetic background, and considerable space and expense of housing canines make them not ideal for use as a model (217). Rodents, a common laboratory model, do not get spontaneous prostate cancer with the exception of the Dunning rat model and its derivatives (222, 223). In addition, the prostate organ itself is significantly different between rodents and humans. In humans, the organ is shaped like a walnut and is a single encapsulated body, whereas in rodents there are three distinct and separate lobes (5, 223). The prostates of rodents do not make PSA (218). Of even greater importance are differences in the organization of the epithelial compartment. In humans, as previously discussed, the basal cells form a continuous layer and, along with neuroendocrine cells, are the only epithelial cell in contact with ECM; the secretory cells are not in contact with ECM and reside on top of the basal cells facing the lumen. In contrast, rodent prostates have few basal cells and the secretory cells are in contact with ECM, which means that adhesion signaling 27 may occur in these cells (5). The ECM in rodents is also less tightly organized. Lastly, stromal cells are found throughout the human prostate, but in rodents the stromal cells are found associated tightly with acini (5, 223). The differences in tissue organization and matrix may also allow a more direct interaction of the secretory cells with the ECM and stromal compartment in rodents than what is found in humans. Genetic manipulations of mice, while helpful in determining the molecular events of prostate tumorigenesis, have also failed to produce a satisfactory model of prostate cancer. Genetic knockdown or overexpression of genes known to be lost or gained during prostate cancer progression including AR, ka3.1, PTEN and ka3.1, often result in only hyperplasia or PIN (218, 223, 224). However, mice with prostate specific Myc overexpression, or PTEN loss in combination with p53 or ka3.1 loss, develop invasive carcinoma without metastasis (223, 225-227). Interestingly, the ka3.1;Pten mutant mice develop tumors that express AR, but are androgen-independent (225, 227). The transgenic mouse model TRAMP (transgenic adenocarcinoma mouse prostate) remains one of the few mouse models that exhibit progressive stages of androgen-dependent prostate cancer ranging from PIN to metastatic androgen- independent prostate cancer, although bone metastasis is rare (228-231). However, the TRAMP model and its derivatives rely on prostate specific transgenic expression of the SV40 T and t antigen to suppress p53 and Rb function (229, 231). While p53 and Rb are commonly lost during prostate cancer progression, it occurs only in late-stage disease, and it is not through T or 28 t antigen expression. There are additional T and t antigen targets that are not associated with prostate cancer; large T antigen can inactivate Bub1, a spindle assembly checkpoint protein (232), and small t antigen can modulate the activity of PP2A, which can impact a plethora of cellular signaling pathways including AR (233, 234). While some mouse models recapitulate some features of the human disease, there is some skepticism that they will to be predictive for therapeutic interventions that will work in humans (218, 223, 235, 236). For all cancers, only about 5% of therapeutics successfully tested on mice with cancer, from 1991- 2000, were approved for use in human patients (237). Currently, there is not an inducible model for prostate cancer and only one report looking at the effect of altering gene expression in the stroma, although this along with more elegant compound mutational models are expected to be developed in the next few years (223, 238). Lastly, there is a need for a prostate specific promoter that is androgen independent, so that researches can better elucidate the effect of castration or ADT on prostate cancer in mice, as current prostate specific but androgen-dependent promoters would also be effected. Thus, while mouse models have been informative in characterizing the molecular events required for PIN and prostate tumorigenesis, they have to date been limited in that the rodent prostate is inherently different in its anatomy and morphology, the models rarely metastasize and when they do they rarely go to bone, there is insufficient molecular and biological diversity of models, and the models have failed to be predictive for human therapies. 29 Cell Lines In vitro, there are a limited number of established cell lines due to the difficulty of successfully isolating and culturing human prostate cancer tissue despite repeated numerous attempts (239-242). This may be due to the slow growing nature of prostate cancer (5, 239, 240) and/or a lack of the proper environmental and growth factors in culture. Most in vitro work is based on three cells lines (PC3, DU145, and LNCaP) and their derivatives (for a comprehensive review of all prostate cancer cell lines see 241, 242). These were all originally isolated from metastatic lesions (bone, brain and lymph node respectively) (240); there is no human prostate cancer cell line isolated from a primary tumor. As such, most of the findings in the literature are based on a limited number of cell lines with limited genetic and biological diversity and representative of only metastatic cancer. Furthermore, their relevance in vivo has only been demonstrated in xenograft models and it remains uncertain what correlation exists in human patients. Interestingly, of these three cell lines, PC3 and DU145 cells have lost AR expression and LNCaP cells have a mutated (T877A) AR. While LNCaP cells remain responsive for androgen, the T877A mutation makes AR promiscuous to ligands other than androgen including gluccocorticoids, estrogen, progesterone, and the AR antagonist hydroxyflutamide (243). It has also been reported that all three of these cell lines have been evolving with passage in culture and thus cells from the same cell line may behave differently in different laboratories (218, 244, 245). For example, although it is well established that PC3 and DU145 cells do not express AR, there are some reports and anecdotal 30 stories that suggest early passages of these cells expressed low levels of AR (246-249). In LNCaP cells, IGF represses AR-mediated transcription in early passage cells, whereas IGF stimulates AR-mediated transcription in late passage cells (108). Despite this, there is no requirement for published studies using these cells to Include a standardized characterization (218). Recently, some new metastatic prostate cancer cell lines have become available that express wild type AR including VCaP (250), MDA PCa 2a and MDA PCa 2b (251), and 22Rv1 (252). We are currently characterizing these cells in our lab to examine their integrin 06 expression and adhesion to LM. There is no in vitro model of androgen receptor positive primary prostate cancer cells. Basal epithelial cells in the prostate gland express 0684 and 0381 integrins and adhere to a basement membrane rich in Iaminin 5 (253). When these cells are placed in culture they retain in vitro a majority of the properties seen in vivo, including the ability to secrete and organize their own Iaminin 5-rich matrix (254, 255). Hence, prostate epithelial cells (PECs) cultured from normal human prostate tissue consist primarily of AR-negative basal cells and their transient amplifying derivatives. Furthermore, although normal prostate basal cells can be isolated and cultured in vitro, AR-expressing secretory cells to date cannot. This may due to the post-mitotic status of the cells, or lack of an environmental condition that is required for their survival. 31 Framework of Dissertation To develop effective and low toxicity prostate cancer therapies that are effective even in castrate resistant prostate cancer, it is imperative to understand the molecular mechanisms regulating survival of both normal and prostate cancer cells. However, these mechanisms are poorly understood, in part due to the limitations of current models. Hence the objective of this dissertation was to generate better models in which to study this disease, and to determine how survival of normal prostate and prostate cancer cells is mediated in the context of integrin and AR signaling. My overall original hypothesis was that the interaction of cancer cells with the matrix and the integration of signals from integrins and AR regulates their survival. In normal cells, AR regulates survival. I proposed to investigate this in three specific aims. Specific Aim 1. Identify how integrins mediate survival of PC3 and DU145 prostate cancer cells in vitro. Specific Aim 2. Identify how integrins mediate survival in prostate cancer cells expressing AR in vitro. Specific Aim 3. Identify how integrins mediate survival in primary prostate epithelial cells expressing AR in vitro. Previous studies in our lab have elucidated three integrin-mediated survival pathways in PEC cells, which recapitulate the prostate basal epithelial cell population (187). Survival of PEC cells on LM5 requires 0381, but not 0684, and is dependent on integrin-mediated, ligand-independent activation of the 32 EGF R and the cytoplasmic tyrosine kinase Src, but not PI3K (Figure 2). Integrin- mediated EGF receptor activation supports cell survival by signaling downstream to ERK (Figure 2). Surprisingly, the death induced by inhibition of EGF receptor or Src in normal prostate cells is not mediated through or dependent on caspase activation. The presence of an autophagic pathway regulated by adhesion to matrix prevents the induction of caspases (Figure 2). Suppression of autophagy permits caspase activation and induction of classical apoptosis. Using these pathways as a framework, I first sought to investigate the integrin-regulated survival pathways in prostate tumor cells starting with PC3 and DU145 cells, which do not express AR and are derived from a prostate metastasis, on LM1 and CLI respectively. This established which integrin- mediated signaling pathways regulate survival in the absence of AR (see Chapter 2). To generate a model of castration resistant prostate cancer, AR or AR mutants were re-expressed in PC3 cells. Using this model, I investigated the downstream targets of AR that regulate survival on LM1 independent of PI3K (see Chapter 3). Last, I generated an in vitro differentiation model in which normal human primary prostate basal cells isolated from patients are differentiated into AR-expressing cells that recapitulate many of the characteristics of secretory cells, including loss of matrix adhesion. Using this model, I investigated the requirement of AR and other signaling pathways for cell survival (see Chapter 4). 33 Autophagy - ' I éRKI f‘ r ‘ r "‘ ‘Death ROS and Caspase-lndependent Death FIGURE 2. Model for LM5-mediated survival in PEC cells. Adhesion of growth factor-deprived PECs to LM5 via 0381 and 0684 integrin mediates cell survival by maintaining starvation-induced autophagy. Signaling via 0381 to ERK through EGFR or through Src is also required for cell survival. The PI-3K/Akt pathway is not activated on LM5 and not required for survival. 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Yu HM, Frank DE, Zhang J, You X, Carter WG, Knudsen BS. Basal prostate epithelial cells stimulate the migration of prostate cancer cells. Mol Carcinog 2004 Oct; 41 (2): 85. Gmyrek GA, Walburg M, Webb CP, Yu HM, You X, Vaughan ED, et al. Normal and malignant prostate epithelial cells differ in their response to hepatocyte growth factor/scatter factor. Am J Pathol 2001; 159 (2): 579- 590. 58 CHAPTER TWO: INTEGRIN-MEDIATED SIGNALING IN PROSTATE TUMOR CELLS Portions of this chapter were published separately as: Edick, M.J., Tesfay, L., Lamb, L.E., Knudsen, BS, and Miranti, C.K., Inhibition of Integrin-mediated Crosstalk with Epidermal Growth Factor Receptor/ERK or Src Signaling Pathways in Autophagic Prostate Epithelial Cells Induces Caspase- independent Death. Mol. Biol. Cell, 2007. 18(7): p. 2481-2490 59 INTRODUCTION In vivo, the precise regulation of epithelial cell homeostasis involves interactions between cells and their microenvironment. Cells receive signals from both the extracellular matrix in the basement membrane and soluble factors secreted by the stroma that precisely control the timing of cell division, growth arrest, differentiation, and survival. Integrins on the cell surface that interact with Iaminin (LM) in the extracellular matrix, such as 0381, 0684, and 0681, are critically involved in mediating survival. Genetic loss of LM5, or its receptor subunits, 03, 06 or 84 integrins, in vivo results in epithelial cell detachment and induction of caspase-mediated apoptosis, even in the presence of soluble factors (1, 2). Integrin 81 null embryonic stem cells produce less LM1 (3, 4), suggesting an important relationship between integrin 81 and LM1 specifically. This detachment induced form of apoptosis has been termed anoikis (5). In vitro anoikis can be rescued by expression of an activated form of FAK, Rac, or Akt (6-8), suggesting that integrin-mediated signaling through these molecules is required to maintain cell survival. However, studies in which specific signaling pathways are inhibited while integrins are still engaged suggest alternative pathways, such as Ras/ERK or Jnk, are required for integrin-mediated survival. Whether signaling from multiple pathways is involved in mediating integrin- dependent survival and whether different pathways are unique to specific cell types has not been extensively investigated. Anoikis has been observed to result in caspase-mediated apoptosis (9). Disruption of the mitochondria, subsequent release of cytochrome c and 60 activation of caspases are hallmarks of classical apoptosis (10) and caspase 3 is required for mitochondria-regulated apoptosis (11). Members of the Bcl-2 protein family are important regulators of mitochondria integrity (10). In addition to classical caspase-mediated apoptosis, several other mechanisms of cell death have been described. Other forms of cell death include caspase-independent cell death, autophagy, or cornification. The role of integrins in regulating cell survival through suppression of these other death pathways is unknown. However, some of the same integrin-induced signal transduction pathways that have been linked to survival are also important for regulating these alternative cell death pathways. For example the Ras/ERK and PI3K pathways act as positive and negative regulators, respectively, of autophagy in several cell types (12). Additionally, epithelial cells have been shown to undergo death by cornification in response to inhibition of ERK and Jnk, but not PI3K (13). Finally, death induced by over expression of Ras, or suppression of Raf in melanoma cells leads to caspase-independent cell death (14, 15). Whether integrin-induced activation of specific signaling pathways plays a role in regulating any of these cell death mechanisms, has not been determined. While studies with various established cell lines have been extremely useful for elucidating potential signaling pathways involved in integrin-mediated survival, it is important to place the findings in the context of a defined organ system where the specific cell type, the integrins expressed, and the matrix being studied are better defined. 61 h I Our work and that of others have demonstrated that integrin engagement is sufficient to activate receptor tyrosine kinases (16-23). We demonstrated that adhesion of normal epithelial cells to matrix is sufficient to induce activation of the epidermal growth factor receptor (EGFR), independently of ligand (18). In addition, we demonstrated that integrin-mediated activation of a subset of signaling pathways, namely the Ras/ERK and Pl3K/Akt pathways, are dependent on integrin-induced EGFR activation. Src kinases have been implicated in regulating integrin-mediated activation of receptor tyrosine kinases (19, 24). Our lab has additionally demonstrated that adhesion of growth factor starved normal primary prostate epithelial cells (PECs) to LM5-rich matrix mediates survival via integrin 0381 through two independent signaling pathways; 1) integrin mediated activation of EGFR and subsequent signaling to ERK and 2) integrin-mediated activation of Src, the former being dependent on 0381 integrin. Interestingly, there was no activation of the PI3K/Akt signaling pathway in PECs on LM5; consequently there was no dependence on this pathway for survival in normal PECs. Inhibition of EGFR/ERK or Src is sufficient to induce cell death, but this death is mediated through a caspase-independent mechanism that is dependent on the generation of reactive oxygen species. During the course of these studies we also discovered that adhesion of PECs to LM5-rich matrix is required to maintain autophagy (refer to Chapter 1, Figure 2) (25). Signaling through 0381, and to a lesser extent 0684, is required for autophagy. Disruption of autophagy, pharmacologically or by blocking 0381, leads to caspase activation and death. 62 Since our studies demonstrated the importance of these pathways in regulating integrin-mediated survival, we set out to use them as a framework to investigate the integrin-mediated survival pathways in the prostate tumor cell lines. Most prostate tumor studies have been done in three metastatic cell lines, PC3, DU145, and LNCaP due to the lack of a human primary prostate tumor cell line. LNCaP and PC3 cells both lack the PI3K/Akt inhibitor Pten; however, LNCaP cells retain wild-type p53 and have a mutated androgen receptor (AR), while PC3 cells have neither (26-28). DU145 cells also do not express p53 or AR, but still retain some Pten expression (26-28). During prostate cancer progression, Iaminin (LM) and collagen (CL) are the predominant matrices observed (29). However, how integrin-mediated adhesion can regulate survival in these cell lines and on these matrices has not yet been determined. We chose to investigate integrin-mediated signaling in the PC3 and DU145 cell lines because they express the proper integrins to adhere and bind to laminin and collagen matrix respectively, and represent two different oncogenic profiles. Although these cell lines do not express AR and the ultimate goal of the thesis was to determine how AR and integrins regulate survival in tumor cells, we first sought to establish what integrin signaling pathways regulated survival in these cells. We hypothesized that the prostate tumor cell line PC3, which has lost expression of PTEN, would be dependent on PI3K signaling for survival. These cells have decreased expression of EGFR, so we hypothesized that EGFR signaling would not be required for cell survival, although signaling through Src 63 may still be important in promoting cell survival. In contrast the cell line DU145, which still express some Pten and EGFR, signaling via Ras/ERK could be mediated through integrin-dependent activation of EGFR. These cells may not be dependent on Pl3K signaling since they still express Pten. To test these hypotheses, we assessed the ability of PC3 and DU145, adherent to either LM1 or CLI matrix respectively, to survive in the context of EGFR, PI3K, Src and downstream signaling inhibitors. These matrices where investigated given their importance in prostate cancer progression. Primary and lymph node metastatic prostate cancer predominately express Iaminin integrin 0681 and it is hypothesized that prostate cancer escapes the gland in part by Iaminin-coated nerves (29-37). The primary metastatic site for prostate cancer is bone, where the microenvironment is rich in collagen, and to a lesser extent Iaminin (38, 39). Although PC3 cells can bind both matrices, we choose to investigate their adhesion to LM1 since this is the preferred substrate for integrin 0681, which is critical in disease progression. Lastly, DU145 integrin-mediated adhesion was investigated on CL1, their preferential matrix. RESULTS In our studies we observed that the relative expression level of EGFR across prostate cancer cell lines and PECs varies (Figure 3A). Considering that EGFR expression level in PC3 cells is lower than the expression level in PECs (Figure 3A), we sought to determine if this prostate tumor cell line maintained EGFR-dependence for integrin mediated survival. We found that adhesion of 64 FIGURE 3. Integrin-mediated signaling on LM1 in PC33. A) 50 pg of whole cell extracts from PECBI, LNCaP, PC3, and DU145 cells were analyzed for EGFR levels by immunoblotting. B-E) Growth factor-starved PC3s were placed in suspension (S) and treated with DMSO (-), 0.5 pM PD168393 (PD), 1 0M AG1478 (AG) prior to plating on LM1 (LM) for 1 hour. In some cases cells were also treated with 10 8M PP2 or 2 0M SU6656. B) EGFR was immunoprecipitated (IP) and levels of tyrosine phosphorylation were monitored by immunoblotting with anti-phosphotyrosine antibodies (pW. C) ERK and D) Akt activation were monitored by immunoblotting of whole cell extracts with the anti-ERK (T 202N204) and anti- Akt (S473) phospho-specific antibodies (pERK Blot, pAkt Blot). Some cells were treated with 10 ng/ml EGF (+EGF) for 10 minutes. E) Src activity was monitored by immunoprecipitation (IP) of p130 Cas and levels of tyrosine phosphorylation were monitored by immunoblotting with anti-phosphotyrosine antibodies (pY). (A-C) Dr. Cindy Miranti, (D,E) Laura Lamb. 65 HGURE3 F Q In M a ‘, U U M ‘- |” 2 0 D 0. .I a. a |-.‘- EGFR LM +EGF EGFRIP: S _ PD AG - pp If“ I I- I- pY I «It... .4... LM +EGF S — PD AG - pD i----’l F‘l - Erk LM +EGF (S - PD AG _ pp I- h - “1|.-I- pAkt l‘f---4l-‘fl-m LM C“ ip‘ . S _ PD LY PP2 su i Ennfir'fl-W l-p130 Cas 66 PC3 cells to Iaminin 5 (or Iaminin 1), does not induce the activation of EGFR or signal downstream to activate ERK (Figure 3 BC; LM1 data not shown). Notably, stimulation of PC3s with EGF induces EGFR and ERK activation, both of which are inhibited by the EGFR inhibitor PD168393, demonstrating that EGFR is functional in these cells (Figure 3B,C). PC3 cells do not express the PI3K/Akt inhibitor Pten, and consequently Akt is activated independent of matrix in PC3 cells, and is not blocked when EGFR signaling is inhibited by PD168393 and AG1478 (Figure 3D). However, this pathway can be activated further by growth factor stimulation, as demonstrated by EGF stimulation (Figure 3D). Lastly, Src activity as measured by phosphorylation of the downstream effector p130 Cas, was also increased upon adhesion to LM1 (Figure 3E). Inhibition of EGFR or PI3K using pharmalogical inhibitors had no affect on p130 Cas phosphorylation, however the Src specific inhibitors PP2 and SU6656 could partially inhibit this pathway (Figure 3E). - To determine if EGFR, PI3K, and Src pathways were important in PC3 cell survival, these molecules were inhibited using the specific pharmacological inhibitors and cell death was assessed over a 124 hour time course, with maximal death occurring at 72 hours. EGFR is not activated by integrin- mediated adhesion to LM1 (Figure 3B), accordingly inhibition of EGFR does not induce significant cell death in PC3 cells as measured by Annexin V staining (Figure 4A). However, inhibition of the PI3K pathway with LY294002 induced cell death in LM-adherent PC3 cells (Figure 4A). The extent of cell death induced by inhibition of PI3K in PC3 cells was only 40% compared to 85% observed with 67 inhibition of EGFR in PECs suggesting that other mechanisms might be involved in regulating PC3 cell survival on matrix (25). We blocked Src signaling in PC3 cells with either SU6656 or PP2 and found that integrin-mediated survival in PC3 cells, like PECs, is also dependent on Src (Figure 4A). Inhibition of Src resulted in more extensive death than inhibition of PI3K, with >70% of the cells dying. Loss of adhesion has been associated with a specialized form of caspase- dependent apoptosis known as anoikis. To determine if PC3s were dying via caspase-dependent apoptosis, we used an enzyme assay to directly measure caspase activity in dying PC3s. Induction of apoptosis in PC3 cells by treatment with LY294002 to inhibit PI3K activity, or SU6656 or PP2 to inhibit Src activity, induced a 2.5 fold increase in caspase activity (Figure 4B). This activity was inhibited by the caspase inhibitor zVAD. Thus, inhibition of either PI3K or Src, but not EGFR, results in caspase 3 activation and death. Next, we wanted to determine what proteins downstream of Src and PI3K were directly regulating cell survival in the PC3 cells. Src has been reported to maintain the levels of the anti-apoptotic protein Bcl-xL (40). Inhibition of Src with SU6656 or PP2 in PC3 cells resulted in decreased expression of the anti- apoptotic protein Bcl-xL (Figure 4C). PI3K has been reported to promote survival through Akt-dependent phosphorylation and subsequent sequestration of the pro-death protein Bad on Ser136 (41), or by driving the expression of the anti- apoptotic protein Bcl-2 (42). PI3K inhibition decreased Akt-dependent phosphorylation on the pro-apoptotic protein Bad (Figure 4D). However inhibition of PI3K had no effect on Bcl-2 (data not shown) or Bcl-xL (Figure 4C). This is 68 A0100 B-ano- * . - *p=0.027 > . as: o ' ‘ :60 $1.50- §40 £31.00? 520 20.50- “ .2000 so - ‘ o>= OGEDEPPPP .1 can. In <<<< g“ “E 5 mass: +->l-_§§ 94 m c. 0. LM PD LY su p92 LM PD LY su fl ~-- .. .-BcI-xL -" - -— - - -P-Bad a—u—lfl—t—t -tubulin [hag—gn- - — I-Bad FIGURE 4. PC3 cells depend on PI3K and Src signaling for survival. PC3 cells were pretreated with DMSO (DMSO or LM), 0.5 uM PD168393 (PD), 10 8M LY294002 (LY), 2 8M SU6656 (SU), or 10 pM PP2 (PP2). In some cases 20 pM of the caspase inhibitor z-VAD was added at the time of plating as indicated (zVAD). Error bars on all graphs represent standard deviation. A) Cells were analyzed for percent Annexin V staining by FACS 72 hours after plating on Iaminin. n = 4. B) Cells were analyzed for caspase 3 activity. Caspase data are expressed as fold increase in caspase activity over that observed in DMSO— treated cells. n = 3. C) Bcl-xL expression was monitored by immunoblotting of whole cell extracts with the anti-Bcl-xL antibody. Tubulin was used as a loading control. D) Bad phosphorylation was monitored by Immunoblotting of whole cell extracts with the anti-phospho-Ser136 (pBad Blot) Bad antibody. Total Bad expression was used as a loading control. 69 consistent with work done by Bondar and McConkey which also found that Bcl-2 does not regulate cell death in PC3 cells (43). Thus in PC3 cells, Src regulates survival through maintaining elevated levels of Bcl-xL and the PI3K/Akt pathway phosphorylates and inactivates Bad. DU145 cells, another prostate tumor cell line, maintain EGFR expression comparable to PEC cells (Figure 3A). Furthermore, adhesion to CLI matrix induces activation of EGFR and treatment with the EGFR-specific inhibitors PD168393 or AG1478 (not shown) blocked integrin-induced EGFR tyrosine phosphorylation (Figure 5A). Adhesion to CLI also resulted in increased ERK activity; this appeared to be mostly independent of EGFR in that treatment of cells with EGFR inhibitor PD168393 only partially blocked ERK activation (Figure 5B). Inhibition of ERK directly with P098059 or U0126 completely blocked ERK activation (Figure 5B). The PI3K pathway was also activated upon integrin engagement of matrix (Figure 5B). This may be partially dependent on EGFR in that the EGFR inhibitor P0198393 partially blocked Akt phosphorylation (Figure 5B). Src signaling was also activated upon integrin engagement of matrix as measured by phosphorylation of the downstream protein p130Cas, however the pharmalogical inhibitor for Src SU6656 was unable to effectively inhibit Src activity at a variety of concentrations tested (Figure 5C,D). The Src inhibitor PP2 le for Bcl-xL in prostate cells. Previous independent studies have shown thaget effects at the concentrations effective in this assay (Figure 5C). This data suggested that all four pathways, PI3K, EGFR, ERK, and Src, may be important for integrin-mediated survival since they are activated upon adhesion to matrix. 70 FIGURE 5. Integrin-mediated Signaling in DU145 Prostate Tumor Cells. DU145 cells were starved of growth factors for 48 hours, treated with nothing (N), DMSO vehicle (D), 0.5 pM PD168393 (PD1), 10 pM PD98059 (PD9) or U0126 (U0), 10 8M LY494002 (LY), or 2 8M SU6656 (SU) or 10 0M PP2 (PP2) and plated on CLI. Cells in suspension (S) were used as a negative control for integrin engagement. A) Activation of EGFR was monitored by immunoprecipitation of EGFR and immunoblotting of tyrosine phosphorylation levels (pY Blot). B) ERK activation was monitored by immunoblotting total cell lysates with anti-ERK phospho-specific antibody (pERK Blot). Activation of PI3K was monitored by immunoblotting total cell lysates with anti-Akt phospho-specific antibody (pAkt Blot). C-D) Activation of Src was monitored by immunoprecipitation of Gas and immunoblotting of phosphorylation levels (pY Blot). D) DU145 cells were treated with increasing concentrations of SU6656 (SU), ranging from 2 to 200 pM. E) After 72 hours, cells were harvested and stained with fluorescence conjugated Annexin V and RI. and analyzed by FACS. 71 FIGURE 5 EGFR IP: CLI S D P1 P9 LY I" " gilt” CLI ,S N DP1P9UOLY [:2: .-¥ .. q-pErk h - --—-q-El‘k lg '- CID-gfl'flzd-pAkt F; .. as u- m -Akt I“ -tubulin C. Cas IP: CLI D. I“ S D SU PP2 | ...... II” P -— —]-Cas C35 "’3 if ‘7 SU % Annexin V Positive I:§-------I'PY PEHQD-pnu-I-Cas 60- 50- 40- 30- 20‘ 10- DJ D P1 P9 LY STR 72 To determine if integrin regulation of the EGFR, ERK, and PI3K pathways were important for DU145 cell survival, these molecules were inhibited using specific pharmacological inhibitors and apoptosis was then measured using Annexin V and propidium iodide (PI) staining with FACS analysis. Staurosporin (STR) was used as a positive control for inducing cell death. Treatment of DU145 cells with P0198393, P098059, or LY294002 for 72 hours after plating induced cell morphologies suggestive of apoptosis; notably cell condensation and membrane blebbing (data not shown). A 2.6 fold increase in Annexin V positivity was induced in the DU145 cells were treated with the EGFR inhibitor, the ERK inhibitor P098059 induced a 1.8 fold increase, and the PI3K inhibitor induced a 2.2 fold increase in cell death (Figure 5E). Thus, the EGFR, ERK, and PI3K pathways are important in integrin-mediated survival of DU145 cells. DISCUSSION Using two different prostate tumor cell lines, PC3 and DU145, we have identified integrin-mediated signaling pathways whereby adhesion to matrix mediates cell survival (Figure 6). In PC3 cells, survival occurs through PI3K and integrin-mediated activation of Src, but not EGFR. Src regulates survival through maintaining elevated levels of BcI-xL and the PI3K/Akt pathway phosphorylates and inactivates Bad. Inhibition of either pathway results in caspase-dependent cell death (summarized in Figure 6A). In contrast, in DU145 cells, cell survival occurs through integrin-mediated activation of EGF R, ERK, and PI3K 73 FIGURE 6. Models for PC3 and DU145 integrin-mediated survival. A) Survival of PC3 cells adherent to Iaminin 1 requires PI3K signaling and integrin- dependent activation of Src, but not EGFR. Src regulates survival through the pro-survival protein, Bcl-xL and the PI3K pathway phosphorylates and inactivates the pro-death protein Bad. Inhibition of either pathway results in caspase-mediated cell death. B) Survival of DU145 cells adherent to collagen l requires integrin-dependent activation of PI3K, ERK, and EGFR. PI3K activates Akt, while EGFR leads to partial activation of Akt. Integrin adhesion also activates Src. 74 FIGURE 6 PC3 Tumor Cells A. . i E ‘1 ' “ :$*mao| (Akt4 / w. chIA-xL d I" Isa Survive DU145 Tumor Cells ‘Collla ’ . (summarized in Figure 6B). Src signaling is also increased upon adhesion to matrix, however we were unable to determine if it is important in promoting cell survival since the Src specific pharmalogical inhibitors were unable to effectively inhibit Src activity at doses that did not have off-target effects. Future studies using Src specific siRNA need to be done to determine the requirement of Src in survival of DU145 cells. Integrin-mediated transactivation of receptor tyrosine kinases has been widely reported; however, the biological significance of this crosstalk is largely unknown. In this study we demonstrate that EGFR is a critical pathway for integrin-mediated survival in 0U145s, but not PC3s Interestingly, normal PEC cells also are dependent on EGF R for survival. 0U145s are not as invasive and metastatic as PC3 cells, perhaps representing an intermediate phenotype between PEC and PC3 cells, explaining why they may have retained EGFR- dependence. While ERK signaling was also increased upon adhesion in DU145 cells similar to PECs, this was independent of EGFR signaling. However, in DU145 cells, EGFR activation may partially regulate the PI3K/Akt pathway. Similar to this, it has been reported that survival of EGFR over expressing NIH- 3T3 cells on fibronectin required integrin-mediated activation of EGFR, but did not involve signaling to ERK, but rather PI3K (24). Like PECs, integrin-mediated survival of primary keratinocytes on LM5 has been shown to involve EGFR signaling to ERK (44), suggesting that in normal cells integrin activation of EGFR and subsequently ERK is important for cells survival, and that this pathway becomes aberrant during cancer progression. Thus it is interesting to speculate 76 that PC3 cells may have lost the requirement for EGFR for survival by becoming dependent on PI3K signaling due to Pten loss, circumventing the requirement for integrin-mediated activation. However, given that the integrin-mediated survival pathways in PC3 and DU145 cells were studied on different matrixes which engage different integrins, this could also be an explanation as to the differences in signaling observed between the two cell lines. Indeed, in short term 1 hour adhesion assays we have observed that adhesion of PECs to Iaminin 1, but not the Iaminin 5-rich matrix, is sufficient to activate Akt (data not shown), suggesting alternative signaling pathways are activated on other matrices. Interestingly, both PC3 and DU145 cells were dependent on PI3K signaling for cell survival. LNCaP cells, which like PC3 cells also lack Pten, have also been reported to be dependent on PI3K signaling for survival (45); inhibition of Pl3K signaling in LNCaPs results in caspase-dependent cell death (data not shown). However, normal PEC cells are not dependent on PI3K for survival (Figure 2) (25). Many human prostate cancers have reduced levels of the negative PI3K regulator, Pten, and the PI3K/Akt pathway is constitutively activated in those tumors (46-48). However, given that the DU145 cells still express Pten, this suggests that prostate tumor cells can still require this pathway for survival independent of Pten status. This suggests that the PI3K/Akt pathway may be favored in tumor development or progression and may provide a therapeutic target that would be non-toxic to normal prostate basal epithelial cells. However, this requires deeper investigation. 77 Whether integrin-mediated activation of other receptor tyrosine kinases is involved in regulating survival on matrix is not known. However, it was recently demonstrated that ligand independent activation of c-Met in PC3 cells was required for cell survival (49). The specific integrins, matrix, and signaling pathways involved in c-Met-mediated survival are currently unknown. Recent work from our lab has demonstrated that in PECs c-Met regulates survival by stabilizing 0381 integrin via a kinase-independent mechanism (Lia Tesfay, unpublished data). 0381 loss via inhibition of c-Met expression blocks integrin- dependent signaling, which compromises cell viability (Lia Tesfay, unpublished data). Our data indicate that PCS cells, which express low levels of EGFR relative to PECs, do not activate EGFR or ERK upon integrin engagement and do not depend on this pathway for integrin-mediated survival. Instead survival of PC3 cells requires PI3K and Src. c-Met is known to activate these signaling pathways in response to HGF, but whether c-Met participates in integrin- mediated signaling to PI3K or Src in PC3 cells has not been determined. Despite extensive characterization of integrin-mediated signaling molecules involved in regulating cell survival on matrix, including FAK, ILK, Src, p130Cas, Akt, ERK, Jnk, and Rac (6-8, 44, 50-54), little is known about how these molecules link up with the cell death pathways. In PC3 cells we found that loss of signaling through Src results in decreased levels of Bcl-xL and loss of PI3K signaling results in decreased Bad phosphorylation. Early anoikis studies suggested that Bcl-2 was involved in regulating cell survival, because its over expression was sufficient to rescue cell death induced by loss of adhesion (50) 78 and adhesion to matrix in some cells increases Bcl-2 levels (55). However, Bcl-2 expression does not correlate with sensitivity to anoikis in prostate cells (43). More recent studies have linked integrin-mediated survival to Bcl-xL (56). Furthermore, loss of androgen receptor expression in LNCaP cells leads to cell death which is accompanied by a reduction in Bcl-xL expression (57), suggesting an important role for Bcl-xL in prostate cells. Previous independent studies have shown that Bcl-xL can be regulated by signaling through EGFR, integrins, or Src (40, 56, 58). Here we demonstrate that in PC3 cells, Src but not EGFR, regulates BcI-xL. In summary, in the prostate tumor cell line PC3 survival on LM1 occurs through Pl3K and integrin-mediated activation of Src, but not EGFR. Src regulates survival through maintaining elevated levels of the pro-survival protein Bcl-xL and the PI3K/Akt pathway phosphorylates and inactivates the pro- apoptotic protein Bad. Inhibition of either pathway results in caspase-dependent cell death. In comparison in DU145 cells on CLI, integrin-mediated activation of EGFR, ERK, and PI3K promotes cell survival. MATERIALS AND METHODS Antibodies EGFR immunoprecipitating and blocking monoclonal antibodies were purified in the Monoclonal Antibody Core at VARI from hybridoma cells obtained from ATCC (HB-8508). EGFR (Ab12) immunoblotting antibodies were purchased from NeoMarkers. ERK and p130Cas antibodies were purchased from Becton- 79 Dickinson Transduction Labs. Phosphospecific antibodies against ERK1/2 (T 202N204), Akt (S473), Bad (S136) and antibodies to Bcl-2, Bcl-xL, and Bad were purchased from Cell Signaling. The anti-phosphotyrosine monoclonal antibody 4G10 was obtained from Upstate Biotechnology. The Akt antibody was described previously (18). Blocking antibodies for 84 integrin (ASC-8) and 03 integrin (P1B5) were purchased from Chemicon and GoH3 06 integrin antibody was obtained from Becton Dickinson. Cell Culture Primary cultures of human prostate epithelial cells (PEC) were derived from normal human prostatic tissue and cultured as described previously (59). Human samples were obtained after institutional IRB approval. PECs were maintained in Keratinocyte-SFM medium (lnvitrogen) supplemented with bovine pituitary extract and EGF. All experiments were conducted on cells between passages 3 and 5. PC3 and DU145 cells were obtained from American Type Culture Collection (ATCC). PC3 cells were maintained in F12K medium (lnvitrogen) supplemented with 10% fetal bovine serum, 2 mM glutamine, 50 U of penicillin and 50 mg of streptomycin per ml. DU145 cells were maintained in Eagle's Minimum Essential medium (lnvitrogen) supplemented with 10% fetal bovine serum, 2mM glutamine, 50U of penicillin and 50mg of streptomycin/ml. 80 Integrin Signaling Preparation of cells for adhesion to extracellular matrices was carried out as described previously (25, 60). Briefly, cells were growth factor-starved for 48 hours, trypsinized, treated with soybean trypsin inhibitor (lnvitrogen), washed in PBS, and placed in suspension in growth factor-free medium for 30-60 minutes. Cells were then either plated on tissue culture plates blocked with 1% BSA (Sigma) and pre-coated 10 pg/mL natural mouse Iaminin 1 (lnvitrogen) or rat tail collagen 1 (Becton, Dickinson and Company). Similar results were obtained in PC3 cells on Iaminin 1 as on Iaminin 5. Occasionally cells were also treated with 2-10 ng/ml EGF (Upstate). A suspension control was maintained at 37°C. Two hours after plating on matrix cells were lysed either in Triton-X (50 mM Tris pH 7.5, 100 mM NaCl, 0.5 mM EDTA, 1% TritonX-100, 50 mM NaF, 50 mM 8- glycerophosphate, 5mM sodium pyrophosphate, 1 mM Na3VO4, 1 mM PMSF, 100 U/ml aprotinin, 10 pg/ml pepstatin, and 10 pglml leupeptin) or RIPA (10 mM Tris pH 7.2, 158 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% NaDOC, 1% Triton- X100, 1 mM Na3VO4, 1 mM PMSF, 100U ml aprotinin, 10 pg/ml pepstatin, and 10 pg/ml leupeptin) buffers. Pharmacological inhibitors, P0168393, AG1478, LY294002, SU6656, or PP2, purchased from Calbiochem, were added to suspension cells 20 minutes prior to plating on matrix; except for SU6656, which was added 16 hours prior to placing cells in suspension. All working concentrations of the pharmacological inhibitors were determined by titrating to the minimum inhibitor concentration that effectively blocked the target of the pharmacological inhibitor for the duration of our experiments. Inhibitor 81 effectiveness was monitored by Western blotting. Specifically, P0168393 and AG1478 were tested for their ability to inhibit EGFR tyrosine phosphorylation, p130Cas tyrosine phosphorylation (a Src substrate) was used to test SU6656 and PP2, phosphorylation of Akt was used for LY294002, and U0126 was tested against phosphorylated ERK. Titrations were performed for each drug in each cell type. Cell Survival Assays Cells were serum starved for 48 hours and placed in suspension as described above and then plated on 1% BSA-blocked tissue culture plates pre-coated 10 pg/mL natural mouse Iaminin 1 or rat tail collagen 1. Pharmacological inhibitors, 1 pM staurosporine (Promega), 0.5 uM PD168393, 1 pM AG1478, 10 LIM P09809, 10 8M U0126, 10 0M LY294002, 0.5-2 8M SU6656, or 10 8M PP2 were then added. Cells were allowed to adhere for 4 hours and then non-adherent cells were removed and drugs were replaced. Cells were incubated for an additional 72 hours. LY294002 was replenished 48 hours after plating. To assess cell death, cells were stained with Annexin V and propidium iodide using a kit obtained from Molecular Probes (lnvitrogen). Staining was carried out according the supplied protocols. For all staining procedures both attached and floating cells were collected. Attached cells were removed by trypsinization and pooled with floating cells and all cells were washed one time. For Annexin V staining, cells were resuspended in Annexin binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCI, pH 7.4) containing Alexafluor488 conjugated Annexin V 82 and incubated in the dark for 15 minutes. Cells were then washed once with Annexin binding buffer, then incubated in 1 pg/mL propidium iodide and 100 pg/mL RNase A for 5 minutes at room temperature, and washed twice more. Samples were put on ice and immediately analyzed. Extent of staining was monitored by FACS using a FACS Caliber (Becton-Dickinson) and CellQuest version 3.1.3 acquisition and analysis software (Becton-Dickinson). Caspase Activity Assays Caspase 3 and 7 activity in PC3 cells was directly measured using a CaspaseGlo 3/7 kit (Promega) following the manufacturers suggested protocol. For PC3 cells, 10,000 cells/well were plated on 1% BSA blocked 96 well plates pre-coated with 10ug/ml of laminin respectively. Cells were plated in the presence of DMSO, 1 8M staurosporine, 0.5 pM P0168393, 10 0M LY294002, 2 8M SU6656, 10 8M PP2. CaspaseGlo reagent was added at various times after inhibitor treatment and incubated for 1 hour at room temperature in the dark. Relative light intensity was measured in each well using a Fluoroskan Assent FL fluorometer and software (Labsystems). lmmunoprecipitation and Immunoblotting lmmunoprecipitation mixtures containing 500-1000ug protein were incubated with the appropriate antibodies for 3 hours at 4°C with either protein A- or protein G- conjugated agarose beads (Pierce) to capture the complexes. All immunoprecipitated complexes were washed three times with their respective 83 lysis buffer. lmmunoprecipitated samples from adhesion assays were resuspended in 2X SDS sample buffer. In some cases 50-75 pg of total cell lysates were placed directly in 2x SDS sample buffer. All resuspended samples were boiled and subjected to SDS-polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane (PVDF). The PVDF membranes were blocked with 5% BSA in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 2 hours, followed by 2 hour incubation with the appropriate primary antibodies in 5% BSA/TBST. After several washes, blots were incubated with a horseradish peroxidase conjugated secondary antibody (Bio-Rad) for 1 hour in 5% BSA/TBST and visualized with a chemiluminescence reagent and captured by a CCD camera in a Bio-Rad Chemi-Doc Imaging System. Levels of activation, relative to total levels of protein, from blots captured by CCD camera were quantified using Quantity One software (Bio-Rad). Blots were stripped in low-pH 2% SDS at 65°C for 60 minutes, rinsed and reprobed for total levels of protein in the immunoprecipitates or cell lysates. ACKNOWLEDGEMENTS We wish to thank Mat Edick, Veronique Schulz, Matt Van Brocklin, Dr. Kate Eisenmann, and the FACS core at VARI for technical assistance. Special thanks to the laboratories of Developmental Cell Biology, Systems Biology, and Cell Structure and Signal Integration at VARI for their constructive suggestions. Dr. Miranti, 0r. Edick, Ms. Tesfay, and Ms. Lamb are supported by the American Cancer Society (RSG CSM-109378). 0r. Miranti is also supported by the 84 Department of Defense Prostate Cancer Research Program of the Office of Congressionally Directed Medical Research Programs (W81XWH-04-1-0044). Additional support was also provided by the generous gifts of the Van Andel Institute. 85 REFERENCES 1. 10. DiPersio CM, van Der Neut R, Georges-Labouesse E, Kreidberg JA, Sonnenberg A, Hynes R0. 0381 and 0684 integrin receptors for Iaminin-5 are not essential for epidermal morphogenesis and homeostasis during skin development. J Cell Sci 2000; 113 (Pt 17): 3051-3062. Ryan MC, Lee K, Miyashita Y, Carter WG. 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Nat Cell Biol 2002; 4 (4): 83-90. 91 CHAPTER THREE: AR-ENHANCED 0681 INTEGRIN AND BCL-XL EXPRSSION PROMOTES ANDROGEN-INDEPENDENT PROSTATE TUMOR CELL SURVIVAL INDEPENDENTLY OF PI3K SIGNALING 92 INTRODUCTION Prostate cancer is the second highest cancer-related death in American men, resulting in over 28,000 deaths per year (1). While primary prostate cancer can be successfully surgically removed or treated with radiation, metastatic prostate cancer cannot. Since prostate cancer is initially dependent on androgen signaling for proliferation and survival, androgen deprivation therapy, i.e. chemical castration, is the major treatment for metastatic prostate cancer. Although patients initially respond to androgen deprivation therapy, they ultimately relapse with castration-resistant tumors which no longer respond to the anti-androgen therapy, offering little hope for long-term disease-free survival. However, inhibition of androgen receptor (AR) expression or its DNA binding activity, even in castration-resistant cells, inhibits their proliferation and leads to cell death (2-4). This suggests that prostate cancer cells are still dependent on AR for survival, even if the cells are no longer responding to physiological levels of androgen. However, the downstream targets of AR that directly regulate survival in castration-resistant cells are largely unknown. Cell adhesion is necessary for the survival of all epithelial cells; integrins are heterodimeric cell surface receptors that mediate adhesion to extracellular matrix (5, 6). Integrin adhesion to matrix results in subsequent activation of many signaling molecules (5, 6). This integrin signaling through specific pathways is required for cell survival and detachment of cells results in rapid induction of cell death, known as anoikis (7). Integrin signaling through MAPK, PI3K/Akt, Src, Rho GTPases, ILK, and FAK signaling can promote survival 93 through increased expression of the pro-survival proteins Bcl-2, BcI-xL, survivin, and McI-1, and through inhibition of the pro-death proteins Bad, Bak/Bax, Bid, Bit1 and Bim (7-9). Integrin expression and signaling is aberrant in many diseases, including prostate cancer where 0681 is the primary integrin expressed in both primary prostate’cancer and lymph node metastases (10-16). In the normal gland, the basal epithelial cells express several integrins; two in particular, 0684 and 0381, allow the cells to adhere to Iaminin 5 in the matrix and are responsible for promoting basal cell survival (17). Basal cells do not express AR, but differentiate into AR-expressing secretory cells. During this process, the majority of integrins are lost as cells differentiate and detach from the matrix. The exception is 0681, which was recently shown to be expressed in secretory cells (18). Even though the secretory cells express 0681, they do not use this integrin to adhere to matrix. Integrin 0681 is localized to cell-cell junctions in secretory cells and its function there is currently unknown. During prostate cancer development, the basal cells are lost and the secretory cells, expressing both AR and 0681 integrin, now adhere to the matrix. In addition, the matrix is remodeled such that Iaminin 5 is no longer present and Iaminin 10, the preferred substrate of 0681, predominates. Thus, the engagement of 0681 integrin in AR-expressing cells in prostate cancer could provide a new mechanism for prostate cancer cell survival. Furthermore, a few studies suggest that AR could, in part, be responsible for maintaining 06 integrin expressing in cancer cells. AR re-expression in 94 prostate cancer cell lines was reported to alter integrin expression and adhesion to matrix (19-21), and constitutive AR expression in immortalized prostate epithelial cells has been demonstrated to increase integrin 06 (22). In addition, the 06 integrin promoter contains a steroid-response element that is capable of stimulating 06 integrin expression in response to progesterone (23). Phosphoinositide 3-kinase (PI3K) signaling, a common downstream target of both integrin and growth factor receptor signaling, is required for survival of most prostate cancers. The phosphatase and tensin homolog (PTEN), a phosphoinositide phosphatase and negative regulator of Pl3K signaling, is lost in ~30% of clinical prostate cancers and in ~60% of metastatic cancers, resulting in constitutive activation of PI3K signaling (24-26). Akt is a major downstream effector of PI3K signaling and regulates survival through phosphorylation and inhibition of the pro-death proteins Bad, Bax, forkhead box-O transcription factors (FOXO), death-associated protein 3 (DAP3), and pro-caspase 9, through increased expression of the pro-survival protein survivin, and by regulating NF- KB and mTOR signaling (7, 27). While the androgen sensitive prostate cancer cell line LNCaP undergoes cells death within 24 hours of inhibiting PI3K/Akt after pharmalogical inhibition, addition of androgen can rescue survival (28). Furthermore, long term androgen ablation of LNCaPs results in cells resistant to PI3K/Akt inhibition (29). Lastly, in vivo prostate regeneration studies demonstrate that AR and Akt signaling can synergize to promote tumor formation even after androgen ablation (30). This suggests that AR signaling, and in some contexts independent of androgen stimulation, may promote survival 95 independent of PI3K signaling by either regulating the same downstream targets of the PI3K pathway, or through a novel mechanism. Li and colleagues demonstrated that AR can also phosphorylate and inhibit FOXO, thus promoting survival independent of PI3K by regulation of a common downstream target (31). Work by Chen’s laboratory has suggested that the pro-survival protein Bcl-xL can also promote survival independent of PI3K signaling in prostate cancer cells, however the mechanism by which Bcl-xL was regulated independent of serum stimulation remained undetermined (32). Whether integrin and AR signaling crosstalk can promote survival independent of PI3K/Akt signaling remains unknown. In this study, we tested the hypothesis that AR-dependent regulation of 0681 expression in prostate cancer cells promotes survival independent of PI3K signaling. AR was re-expressed in the prostate cancer cell line PC3. Re- expression of AR in PC3 cells lead to increased transcription and expression of integrin 06, and adhesion to Iaminin subsequently increased the activity of PAK1I2 and NF-KB p65, increased transcription and expression of the pro- survival protein Bcl-xL,- and made the cells resistant to cell death induced by PI3K inhibition. Loss of integrin 06, NF-KB p65, or Bcl-xL re-sensitized AR- expressing PC3 cells to PI3K-dependent survival. Treatment of AR expressing PC3 cells with the AR inhibitor RU486 or AR-specific siRNA, or expression of AR mutants that block the ability of AR to translocate to the nucleus (ANLS), but not to bind ligand (ALBD), largely restored the parental PC3 phenotype, including 96 PI3K-dependent survival. These results are largely supported by siRNA knock- down of AR in LNCaP cells. RESULTS The ability to assess the role of 0681 in AR-dependent prostate cancer survival has been hampered by the lack of appropriate cell models that recapitulate the AR/0681 profile seen in vivo. For instance, the most well characterized AR-expressing prostate cancer cell line, LNCaP, expresses 0681 (as well as others), but adheres poorly to Iaminin. On the other hand, the PC3 cell line expresses 0681 and adheres nicely to Iaminin, but does not express AR. Unfortunately, PC3 cells also express a lot of other integrins, including another Iaminin integrin 0381, which is not consistent with what is seen in vivo. Previous studies indicated that AR expression in PC3 cells changes integrin expression (19, 20). Furthermore, both wild type and AR mutants can be expressed in the absence of endogenous AR, which would ordinarily complicate the interpretation of the results. Lastly, any observable phenotypes can be attributed to AR by direct testing with AR siRNA. Viruses were used to introduce empty vectors (PC3-puro or pLKO), sequence-verified wild-type AR, or two AR mutants into PC3 cells. These cell lines were selected and constantly maintained in charcoal-stripped serum to avoid selection against growth suppression, a problematic side-effect of AR expression in PC3 cells (33). AR expression was constantly monitored and only early-passage (< 20) cells were used. To understand which function of AR is 97 important to the observed phenotypes, two well-characterized AR mutants were selected. The ANLS mutant is defective in AR translocation to the nucleus and in DNA binding (34, 35). The ALBD mutant is unable to bind ligand (30). To ensure that AR was not too highly over expressed, stable clonal cell lines that expressed approximately the same level of AR as LNCaP cells were selected. AR expression, in the absence of androgens, was confirmed by immunoblotting and immunoflourescent (IF) staining with anti-AR antibodies (Figure 7A, B). Immunoblotting showed that the wild-type AR clone PC3-AR-1 had comparable expression to LNCaP cells, and clone PC3-AR-2 had higher AR expression (Figure 7A). All the AR mutants had approximately the same level of AR expression as the LNCaP cells (Figure 7A). AR localization was both cytoplasmic and nuclear in wild-type AR expressing clones by IF staining (Figure 7B). Surprisingly, the ALBD mutant was predominately nuclear, while the ANLS mutant was exclusively cytoplasmic as expected (Figure 7B). AR localization was not significantly altered by exogenous treatment with androgens (DHT). To access AR functionality, the expression of known AR-target genes was examined by immunoblotting. PC3-AR-1 and PC3-AR-2 cells expressed higher levels of the AR-target genes ka3.1 and prostate specific antigen (PSA) than the empty vector control cells (Figure 7C), indicating AR is functional. While the full-length androgen-responsive protein TMPRSS2 was expressed in the empty vector PC3- puro cells as well as the AR expressing clones, only the cleaved and activated serine protease domain could be detected in the AR expressing clones (Figure 98 FIGURE 7. On LMI, AR is a survival factor that acts independently of PI3K signaling and androgen. A) AR and tubulin expression levels were monitored by immunoblotting whole cell lysates of LNCaP, PC3 empty-vector (PC3-puro or PC3-pLKO) pools and AR expressing clones (PC3-AR, PC3-ALBD, and PC3- ANLS). B) PC3-puro (PP), PC3-AR-1 (AR), PC3-ALBD-28 (ALBD), and PC3- ANLS-4 (ANLS) cells were immunostained to detect expression of AR (green) and imaged by epifluorescence microscopy. Nuclei (blue) were visualized by Hoechst staining. Clones not shown had similar staining to the representative clones shown. C) Growth factor-starved and charcoal-stripped PC3-puro (PP), PC3-AR-1 (AR1), and PC3-AR-2 (AR2) cells were plated on LMI and treated with vehicle or 10 nM DHT for 72 hours. ka3.1, the full length (FL) and cleaved (SP) forms of TMPRSSZ (TMP), and PSA expression were monitored by immunoblotting. Tubulin served as a loading control. 0) PC3-puro, PC3-AR- 1, PC3-AR-2 cells were growth factor-starved in charcoal-stripped media. Cells were then plated on CLI or LMI in the presence of DMSO or 10 0M LY294002 (LY) to inhibit PI3K signaling, for up to for 4 hours and then non-adherent cells were washed away and drugs and DHT were replaced. Cell viability was measured 72 hours later by fixing and permeabilizing cells for nicked DNA using TUNEL staining and FACS analysis. n = 2. E-H) PC3-puro, PC3-AR-1, PC3- AR-2, PCB-pLKO, PC3-ANLS (NLS), or PC3-ALBD (LBD) cells were growth factor-starved in charcoal-stripped media. Cells were then plated on LMI with vehicle or 10 nM DHT in the presence of DMSO or 10 LIM LY294002 (LY), for up to for 4 hours and then non-adherent cells were washed away and drugs and DHT were replaced. After 72 hours cells were either counted for trypan blue staining E, G-H) or fixed, permeabilized, and stained with propidium iodide (PI) to detect dead cells (sub G1) by FACS analysis ~F) Error bars on all graphs represent standard deviation; n = 3-5. 99 FIGURE 7 A- 3,378 3- PP AR ALBD ANLS '7 0‘ ° ° 6 3 3 a g g g 5 5 3 z z u 9 e <.I <9 <9 <.1 °assasass 5 l a. 0. II. a Q. E L - _ _—‘- -AR IW Tubuun C. D. a O O 8 a 8 fl. .1 I.” Z I? :2 PC3-puro PC3-AR-1 PC3-AR-2 E. F. a ,3 8° ' nomso . U a ‘- _ o 40 - “g .n 1 32°- E == 0. at PC3-puro G H. 80 IDMSO lLY 100 % .3 so ‘3 8 so 3 O E 2 40 i '2 20 E' a 0 " E s at 100 7C). Addition of androgen (DHT) did not significantly alter expression of ka3.1, PSA, or cleaved TMPRSSZ, presumably because AR nuclear localization occurs independently of ligand in these cells (Figure 7B). Inhibition of PI3K signaling in PC3 cells with the pharmalogical inhibitor LY294002 results in cell death (36). To determine if the integration of integrin signaling and AR expression could protect cells from death when PI3K signaling was inhibited, serum-starved AR-expressing clones were plated on Iaminin 1 (LM1), a preferred substrate for 0681 integrin, or the 0281 substrate, collagen l (CLI) in the presence or absence of LY294002, and analyzed over a 72-hour time course. Cell death was measured by Terminal Deoxynucleotide Transferase dUTP Nick End Labeling (TUNEL) staining and FACS analysis. Inhibition of PI3K signaling with LY294002 resulted in maximal death at 72 hours in the PC3- puro cells, but only when plated on LM1 (Figure 7D). In contrast, the AR- expressing clones did not experience significant cell death from LY294002 treatment on LM1 or CLI (Figure 70). Thus, in the context of adhesion to LM1, but not CLI, AR contributes significantly to cell survival, since LY294002 was sufficient to induce death in the PC3-puro cells but not in the AR expressing cells. Treatment of PC3-AR-1 or PC3-AR-2 cells adherent to LM1 with LY294002 also failed induce cell death as measured by trypan blue staining or propidium iodide (PI) staining of sub G1 cells (Figure 7E-F). All the observed effects were independent of androgen (DHT). This difference in survival was not strictly due to cell cycle status since PC3-AR-1 cells grow at the same rate, whilePC3-AR-2 cells grow slower than PC3-puro cells (data not shown). 101 Furthermore, the ability of AR-expressing cells to survive on LM1 was not due to AR-mediated hyper-activation of the PI3K/Akt pathway and LY294002 was still a potent inhibitor of Pl3K/Akt signaling in the AR expressing cells (data not shown). The AR nuclear localization mutant ANLS (Figure 7G), but not the AR ligand binding mutant ALBD (Figure 7H), restored sensitivity to PI3K inhibition resulting in cell death. Thus, AR can promote survival independent of PI3K signaling in PC3 cells plated on LM1. This requires AR localization to the nucleus, presumably to bind DNA. Expression of AR in PC3 cells was previously shown to decrease 0684, 0381, and 0281 expression (19, 20). On the other hand, expression of AR in DU145 cells decreased 0684, but increased 0281 (21). Since different matrixes engage different integrins and the dependence on P|3K signaling was only observed when the cells were cultured on LM1 but not CLI (Figure 70), we sought to determine if AR affected integrin expression in cells plated on LM1. Integrin surface expression was analyzed by FACS in the empty vector PC3 cells and compared to the AR-expressing PC3 cells after adhesion to LM1. AR, independent of DHT, reduced expression of integrins 03 2-3 fold and 05 3-6 fold, but increased expression of integrin 06 3-5 fold (Figure 8A). AR had relatively little or no effect on integrin 02 and there was a slight decrease (<2-fold) in integrin 81 (Figure 8A). Integrin 84 levels were very low in PC3-puro cells (mean fluorescent values 18.05 -/+1.73) and did not significantly change upon AR expression (mean fluorescent value 13.82 -/+ 6.49 and 10.18 -/+ 2.79 for PC3- AR-1 and PC3-AR-2 respectively). Since integrins must be in heterodimeric 102 pairs to be expressed on the cell surface and there was a decrease in the 03 and 05 subunits and an increase in 06, there must be an overall decrease in integrins 0381and 0581 and an increase in 0681 since integrin 84 levels did not increase to compensate for the large increase in 06 surface expression. During prostate cancer development there is a loss of most integrins and an increase in expression of integrin 0681 (10-16), suggesting that our AR-expressing PC3 cells recapitulate some of what is seen in vivo. To verify that AR can regulate integrin 06 eXpression in other cell lines, endogenous AR in LNCaP cells was knocked- down by transfection with AR-specific siRNAs. After 48 hours, AR levels were greatly diminished as observed by immunoblotting (Figure 8B). This was accompanied by a ~12% decrease in integrin 06 expression as measured by FACS (Figure BC). Since integrin-mediated adhesion can promote cell survival and there was an increase in integrin 06 with AR expression, we sought to test the hypothesis that AR was promoting survival through up-regulation of integrin 06. To test this, integrin 06 expression in the AR expressing clones were decreased as close as possible to empty vector levels by careful titration of integrin 06 siRNA (Figure 80). A non-specific siRNA (scram) was used as a control in these and all subsequent experiments. Loss of integrin 06 did not have a significant or consistent affect on AR expression, indicating that integrin 06 is downstream of AR (Figure 80). Cells were treated with integrin 06 siRNA or scram for 72 hours to induce partial knock-down of integrin 06, then cells were plated on LM1 and treated with LY294002. After 72 hours, cell viability was assessed by trypan blue 103 staining. A 63-73% reduction in 06 integrin expression in the presence of LY294002 is sufficient to induce cell death in AR expressing cells (Figure 8E). These data indicate that AR is promoting survival through integrin 06. To verify that these effects were due to AR expression and not clonal selection, AR expression was knocked in the AR expressing cells using siRNA prior to treatment with the PI3K inhibitor, LY294002 (Figure 8F,G). Loss of AR in AR expressing clones lead to decreased integrin 06 expression by immunoblotting (Figure 8F) and increased cell death when the cells were treated with LY294002 (Figure 8G). Overall, these data indicate that AR is a pro-survival factor in PC3 cells that acts independently of PI3K signaling through increased integrin 06 expression. We then wanted to determine the mechanism by which AR and integrin 06 regulate survival independent of PI3K signaling. We first tested the hypothesis that ARfIntegrin 06 was regulating a common downstream target of PI3K/Akt signaling. The PI3K/Akt pathway has been demonstrated to promote survival through several mechanisms, including regulation of the pro-death proteins Bad or FOXO (36-39) and the pro-survival proteins survivin and Bcl-2 (40-42). Furthermore, these proteins have also been reported to be regulated by AR (38, 40, 41, 43) or integrins (44, 45). Bcl-2 and survivin have also been associated with prostate cancer progression (46-49). However, in the AR expressing PC3 clones, there was no decrease in Bad or FOXO activity and no increase in survivin or Bcl-2 levels compared to empty vector control cells, with or without DHT or plating on LM1 or CLI matrix (data not shown). Together, these data 104 FIGURE 8. AR promotes survival through up-regulation of integrin 06. A) PC3-puro, PC3-AR-1, PC3-AR-2 cells were growth factor-starved in charcoal- stripped media and plated on LMI in the presence of vehicle or DHT. After 72 hours, cells were treated with flourescent-conjugated antibodies against the indicated integrins and analyzed by FACS. Mouse and Rat lgG were negative controls. lgG controls were subtracted from mean flourescent values then values for AR expressing cells were normalized to those of PC3-puro cells. Error bars represent standard error; n = 5-8. B-C) LNCaP cells were treated with siRNA against AR (siAR) or non-specific sequence (scr) for 72 hours. B) LNCaP lysates were immunoblotted to monitor AR expression. Tubulin was used as a loading control. C) LNCaP cells were treated with flourescent- conjugated antibody against integrin 06 and analyzed by FACS. Rat IgG was the negative controls. Values given are for mean fiourescent values minus IgG control. Error bars represent standard error; n = 2. D-G) PC3-puro (PP, Puro), PC3-AR-1 (AR1), and PC3-AR-2 (AR2) cells were treated with siRNA against integrin 06 (siA6), AR (siAR) or non-specific sequence (scr) for 72 hours and plated on LMI. D, F) Integrin 06 (ITGA6) and AR expression was monitored by immunoblotting. Tubulin was used as a loading control. E, G) Cells were treated with DMSO or LY294002 (LY) for 72 hours. Cell viability was determined using trypan blue staining. Error bars represent standard deviation; n = 3. (A, D-G) Laura Lamb, (B) Jelani Zarif, (C) Laura Lamb and Jelani Zarif, 105 PF 03 AR1 ' lPC3-AR-1 oI-IT- - IPC3-AR-1 DHT+ I PC3-AR-2 DHT- . IPC3-AR-2 DHT-I» 05 06 AR2 b1 scr sIA6 scr 3M6 scr siA6 - --. - --|-ITGA6 ---—|-AR [-----]-tubulin F. PP AR1 AR2 , scr siAR scr siAR scr siAR l - - | - --- u- -AR -ITGA6 -tubulln % Trypan Blue Gels ' % Trypan am can {.5 FIGURE 8 i 1 l oBSSB scr siAR IDMSOILY 106 scr siAR scr siAR scr siAR PC3-pure PC3-AR-1 PC3-AR-2 suggest that in AR expressing PC3 cells Bad, FOXO, survivin, or Bcl-2 is not the downstream target of integrin- and AR-signaling. The pro-survival protein Bcl-xL has been reported to promote survival independent of PI3K signaling in prostate cancer cells (32), and we previously demonstrated integrin-mediated adhesion to LM1 in PC3 cells led to increase BcI-xL expression through the non-receptor tyrosine kinase Src (see Chapter 2) (36). Increased Bcl-xL expression is also associated with prostate cancer progression (46, 50, 51). Immunoblotting of total cell lysates demonstrated that Bcl-xL is up-regulated in AR expressing PC3 cells (Figure 9A), suggesting BcI-xL could be the mechanism by which AR promotes survival independent of PI3K signaling. To demonstrate that up-regulation of Bcl-xL is due to AR expression, AR-expressing cells were treated with AR siRNA and expression of BcI-xL was monitored. In collaboration with Jelani Zarif, we demonstrated that loss of AR in AR expressing clones resulted in down-regulation of both integrin 06 and Bcl-xL (Figure 9B). To determine if Bcl-xL expression is dependent on integrin 06, integrin 06 expression was decreased using integrin 06 siRNA. Decreased integrin 06 resulted in decreased Bcl-xL (Figure 9C). Lastly, we demonstrated that loss of AR in LNCaP cells also resulted in a modest decrease in both integrin 06 and Bcl-xL levels. Together, these data indicate that AR, acting via integrin 06, drives increased expression of the pro-survival protein Bcl-xL. To determine if BcI-xL is required for Pl3K-independnet survival, Bcl-xL was knocked down in the AR expressing cells to the approximate levels found in 107 A. B. AR1 AR2 PP AR1AR2 scr sIAR scr sIAR ‘ -AR -ITGA6 -Bcl-xL 1-tubulin C- PP AR1 AR2 D- LNCaP scr siA6 scr slAs scr siA6 36' SIAR — - — c- - -AR -- -- «II- -ITGA6 FIGURE 9. AR and integrin 06 regulate Bcl-xL expression. PCS-pure (PP), PC3-AR-1 (AR1), PC3-AR-2 (AR2) and LNCaP cells were treated with siRNA against AR (siAR), integrin 06 (siA6), or non-specific sequence (scr) for 72 hours. AR, integrin 06 (ITGA6), and Bcl-xL levels were monitored by immunoblotting of whole cell extracts using AR, integrin 06 (ITGA6), and BcI-xL specific antibodies. Total levels of protein were monitored by immunoblotting with anti-tubulin. (A, C) Laura Lamb, (B, D) Jelani Zarif. 108 the PC3-empty control cells using Bcl-xL siRNA (Figure 10A). Partial loss of Bcl- xL in the AR expressing clones in the presence of LY294002 induced cell death to the levels found in the PC3-empty cells treated with LY294002 (Figure 10B). Complete loss of Bcl-xL resulted in complete loss of viability (data not shown). To demonstrate that Bcl-xL over-expression is sufficient to promote survival independent of PI3K signaling, retroviruses were used to infect cells with an empty vector or a vector expressing Bcl-xL and stable clonal cell lines were selected. BcI-xL over-expression to the levels found in AR expressing clones was confirmed by immunoblotting (Figure 10C). As expected, Bcl-xL over expression did not result in AR expression or changes in integrin expression (Figure 10C, data not shown). Moreover, Bcl-xL over-expressing cells did not die when treated with LY294002 (Figure 100). Thus, Bcl-xL is a potent pro-survival factor and can promote survival independent of PI3K signaling. We previously demonstrated integrin-mediated adhesion to LM1 in PC3 cells led to increased Bcl-xL expression through the non-receptor tyrosine kinase Src (see Chapter 2) (36). AR has also been reported to promote Src activity though its proline-rich region, which is capable of binding the SH3 domain of Src (52). We sought to determine if increased Src activity was responsible for the increased Bcl-xL expression. Src is activated by phosphorylation at Y416 and inhibited by phosphorylation at Y527 (53). Therefore, Src expression and activity was measured by immunoprecipitation of Src and immunoblotting for phosphorylation of Y416, dephosphorylation of Y527, and total Src. There was increased Src expression and activity in the AR expressing clones (Figure 11A). 109 A' B. 80 a IDMSO PP AR1 AR2 % 60 (oer eI-xL ecr sI-xL scr sI-xl. o ‘ I -n - . aJ-BcI-xL 3 40 - : ’ ~ In .. - 1- . 20 i g 22 MAR a - W _ 0 . i--- - I I. .l-tubulln E scr scr sl-xL scr sl-xL pp AR1 3‘ Pure PC3-AR-1 PC3-AR-2 scr ecr sI-xL I - ' Bel-XL c. '7 '9 '7 D- . E E 5:"? ii :3 i ----fl'3°"’¢ £403 I -v- ‘AR i20- P.‘ 0 ‘ [._----l-tubulln ; Puro BclxI-1BclxléBCIXI-7 FIGURE 10. BcI-xL promotes survival independent of PI3K signaling. A) Cells were treated with siRNA specific against BcI-xL (si-xL) or non-specific sequence (scr) for 72 hours. BcI-xL and AR levels were monitored by immunoblotting of whole cell extracts using BcI-xL and AR specific antibodies. Total levels of protein in the lysates were monitored by immunoblotting with anti- tubulin. Treatment of PC3-AR-1 cells with siRNA for more than one experiment is shown. B) Cells were then plated on LMI and treated with DMSO or LY294002 (LY) to inhibit PI3K signaling. Cell viability was measured 72 hours after drug addition by trypan blue staining. Error bars represent standard deviation; n = 3. C) PC3 cells were made to over express BcI-xL by infecting cells with a retrovirus containing an empty or BcI-xL construct (PP and Bcl-xl respectively) and stable clones were selected in puromycin. D) PCS-puro (puro) and PC3-BcI-xl (Bclxl) clones were plated on LMI and treated with DMSO or LY294002 (LY) for 72 hours. Cell viability was then measured by trypan blue staining. Error bars represent standard deviation; n = 3. 110 In collaboration with Jelani Zarif, we demonstrated that while loss of AR expression in these cells results in partial loss of Src activity it had no effect on Src expression (Figure 11B). In LNCaP cells, AR knock-down of AR also leads to a partial loss in Src activity and there is a slight decrease in total Src levels (Figure 11C). To test whether integrin 06 signaling was enhancing Src activity, integrin 06 expression was knocked down to PC3-puro levels using integrin 06 siRNA. Partial loss of integrin 06 failed to affect Src activity, suggesting that AR- enhanced Src activity was not downstream of integrin 06 (Figure 110). In PC3- AR clones, partial knock down of Src also does not lead to decreased levels of Bcl-xL or AR (Figure 11E). Lastly, while knock down of Src in PC3-puro cells resulted in a two-fold increase in cell death, it had no significant affect on cell survival in the AR expressing cells (Figure 11F). These data indicate that AR expression alters Src activity, this dramatic change in Src activity is not involved in the integrin 06/Bcl-xL signaling axis nor is it required for survival. In addition to promoting cell survival, Src has been well-characterized in promoting normal and cancer cell migration and invasion (54). AR expressing clones have a different morphology and display more filopodia than the PC3-puro cells as observed by immunoflourescent microscopy (Figure 12 AB). Filopodia formation is associated with a more migratory phenotype (55). Correspondingly, the AR expressing clones are more migratory than the PC3-puro cells as determined by a Boyden chamber migration assay using LM1 as a chemoattractant (Figure 12 C, 0). Work by Jelani Zarif has also demonstrated that the AR expressing clones are more invasive than PC3-puro cells, and that 111 A- PP AR1 AR2 B. AR1 AR2 .p.y415 scr siAR scr siAR Iii-mu —.-... smp 6_‘= Src lP c. LNCaP D. pP AR1 AR2 scr “AR scr siA6 acr siA6 scr siA6 r, d. - a-e -P-Y416 meet-Y527 ————4—— '- ug - - -deP-Y527 Src IP ‘ Q .1— «Src SrcIP - .... Ii -ITGA6 ------ -tubulln WCL E. PP AR1 AR2 F- 30 lscram scr siSrc ecr siSrc scr siSrc § 25 lslSrc i - a - " i '3": g 20 SrclP a 15 -- - nl-Bcl-xL g 10 . a. :EEEEI I“ E 5 Q-p-n-l-tubull" 3‘ 0 WCL PC3-puro PC3-AR-1 PC3-AR-2 FIGURE 11. AR regulates Src activity. A-D) PC3-puro (PP), PC3-AR-1 (AR1), PC3-AR-2 (AR2), or LNCaP cells were treated with AR siRNA (siAR), integrin 06 siRNA (siA6) or a non-specific sequence (scr) for 144 B) or 72 C-0) hours. Src was then immunoprecipitated (IP) and activity was measured by immunoblotting for phosphorylation of tyrosine 416 (pY416) and loss of phosphorylation on tyrosine 527 (deP-Y527). Total Src expression was also monitored. In some cases, integrin 06 (ITGA6) and tubulin expression was measured by immunoblotting of whole cell lysates (WCL). E) Cells were treated with Src siRNA (siSrc) or a non-specific sequence (scr) for 72 hours, then Src activity was measured as in A-D. Whole cell lysates (WCL) were also measured for BcI-xL and AR expression by Immunoblotting. Tubulin was used as a control for loading. F) PCS-puro, PC3-AR-1, and PC3-AR-2 cells were plated on LMI and treated with Src siRNA (siSrc) or a non-specific sequence (scr) for 72 hours. Cell viability was measured by trypan blue staining. Error bars represent standard deviation; n = 3. (A, D-F) Laura Lamb, (B-C) Jelani Zarif. 112 A. B. Pcs- PC3-AR-1 PCS-AR-Z “'° 100% ' _ 80% - 2 g 60% -' 3‘ 40% i F-Actin 20% t Vinculln DNA 0% ‘ PP AR1 AR2 I >50% of cell covered by filopodia <50% of cell covered by filopodia I No filopodia c. 0. 250 A 0.250 I.» E g 200 § 0.200 3 150 0.150 E .5 2100 3 0.100 ’ 50 at 0.050 D 0 O 0.000 PP AR1 AR2 PP AR1 AR2 FIGURE 12. AR promotes filopodia formation and migration. A) Cells were plated on LMI for 1 hour then fixed, permeabilized, and stained for F-actin (red), vinculin to visualize focal adhesions (green specks), and nuclei (blue). B) Quantification of the presence of filopodia on cells. 100 cells were counted per experiment; n = 4. C, D) The ability of cells to migrate was tested using a Boyden-chamber migration assay with LMI gradient as the chemoattractant. Cells that migrated to the bottom chamber were then stained with crystal violet and (C) counted or (0) crystal violet staining was eluted from cells and quantified by 00 reading. Error bars represent standard deviation; n = 3. 113 knockdown of Src or AR in the AR expressing clones significantly decreases their invasion (data not shown). AR is a transcription factor whose activity depends on nuclear localization and expression of the AR ANLS mutant that is unable to translocate into the nucleus and bind DNA, was also unable to protect cells from LY294002-induced death (see Fig 76). To determine if AR expression was regulating integrin 06 and Bcl-xL transcription, RNA was isolated from PC3-puro and AR expressing clones, reverse transcribed and quantitative RT-PCR (qRT-PCR) was preformed. There Was over a 10-fold increase in integrin 06 and Bcl-xL mRNA levels compared to PC3-puro cells, independent of DHT addition (Figure 13A,B). The AR increase in BcI-xL mRNA is in agreement with studies in LNCaP cells where treatment with androgen or AR-specific siRNA leads to a respective increase or decrease in Bcl-xL mRNA (2, 51). In collaboration with Jelani Zarif, to verify that this was an AR-dependent effect, PC3-AR-1 cells were treated with the anti- androgen RU486 or AR-specific siRNA. RU486 is reported to recruit co- repressors to AR transcriptional complexes thereby inhibiting AR-mediated transcription (56). RU486 decreased integrin 06 mRNA expression as measured by RT-PCR, (Figure 13C). RU486 treatment also resulted in a decrease in the protein levels of integrin 06 expression with approximately the same severity as AR-specific siRNA treatment (Figure 130). There was also a decrease in integrin 81 (Figure 130). Since integrins must be expressed as heterodimers in order to be stably expressed, loss of the integrin 06 binding partner of integrin 81 may be leading to its degradation. Since AR must be in the nucleus to act as a 114 FIGURE 13. AR regulates integrin 06 and BcI-xL mRNA expression. A-B) PC3-puro (PP), PC3-AR-1 (AR1), and PC3-AR-2 (AR2) cells were plated on LMI and treated with vehicle (ethanol) or DHT for 72 hours. mRNA was then isolated and reverse transcribed. Integrin 06 (ITGA6) A) or Bcl-xL B) mRNA expression was measured by qRT-PCR. Gene expression was normalized to 18s rRNA then expressed as fold change relative to vehicle-treated PC3-puro cells. Error bars represent standard deviation; n = 3. C) PC3-puro (PP) and PC3-AR-1 (AR1) were plated on LM1 then treated with DMSO (0), PBS (P), or RU486 (RU) for 72 hours. mRNA was then isolated and RT-PCR was preformed to measure integrin 06 (ITGA6) levels. GAPDH expression was used as a loading control. 0) PC3-puro (PP) and PC3-AR-1 (AR1) were plated on LM1 then treated with DMSO (0), RU486 (RU), AR siRNA (siAR), or non-targeting siRNA (scr)_for 72 hours. Cells were lysed and immunobloted to monitor integrin 06 (ITGA6) and 81 (ITGB1) expression. Total levels of protein were monitored by immunoblotting with anti-tubulin. E) PC3-pLKO (PL) and PC3-ANLS-AR (ANLS) clone lysates were monitored for integrin 06 (ITGA6) and Bcl-xL expression by immunoblotting. Tubulin expression was used as a loading control. F) PC3-puro, PC3-AR-1 (AR1), PC3-AR-2 (AR2), PC3-pLKO, PC3-ANLS-AR-4, and PC3-ANLS-AR-30 cells were growth factor-starved in charcoal-stripped media and plated on LMI. After 72 hours, cells were treated with flourescent—conjugated integrin 06 antibody and analyzed by FACS. Rat IgG controls were subtracted from mean flourescent values then values for AR expressing cells were normalized to those of the corresponding vector cells. Error bars represent standard error; n = 2. G) LNCaP cells were serum- starved in charcoal-stripped media for 48 hours, then treated with vehicle (veh), DHT, or R1881 for 24 hours. mRNA was then isolated and reverse transcribed. integrin 06 (ITGA6), Bcl-xL, and PSA mRNA expression was measured by qRT-PCR. Gene expression was normalized to 18s rRNA then expressed as fold change relative to untreated cells. Error bars represent standard deviation; n = 1. (A-B, E-G) Laura Lamb, (C-D) Jelani Zarif. 115 A N 01 O I I 1 Fold Chance mus mRNA P A O M O .0 ANLS F- PL 4 30 I- - -|-ITGAB FIGURE 13 Fold Change Bcl-xL mRNA 9' AR1 0- PP iscr RU D screIAR RU 3 PC3-puro PC3-AR-1 PC3-AR-2 AR1 -- - - Tl-ITGAB I .i.‘ «I: l-ITGB1 I .”- l-tubuln l I— - ‘I-Tubulln d or I Fold Chance mRNA .0 N O O O ON-DO’ Protein Over Vector Fold Chnage In IT GAB LNCaP I ITGA6 I Bcl-xL I PSA veh DHT R1881 116 AR1 AR2 NLS4 NLS30 transcription factor, we tested the affect of expression of the AR ANLS mutant on integrin 06 and BcI-xL expression. Expression of the ANLS AR mutant in PC3 cells did not result in increased integrin 06 expression compared to empty vector PC3-pLKO cells (Figure 13E,F). Lastly, stimulation of LNCaP cells for as little as 24 hours with DHT or the more potent synthetic androgen R1881 results in increased integrin 06 and Bcl-xL mRNA expression as determined by qRT-PCR. PSA was used as a positive control. Together, this suggests that AR transcriptionally regulates integrin 06 and Bcl-xL expression. It still needs to be determined if the effect of AR on 06 integrin is direct or indirect. The effect of AR on BcI-xL expression is indirect, since we demonstrated that 06 is required for Bcl-xL expression (see Figure 80). We sought to determine how integrin 06 could drive Bcl-xL expression, since it was not through Src (Figure 11E). The transcription factor NF-KB (RelA) has been reported to directly bind the promoter of and drive transcription of Bcl-xL (57, 58). Furthermore, increased NF-KB activity is associated with prostate cancer progression (59), castration-resistance (60, 61), poor prognosis (62, 63), biochemical failure (i.e. PSA relapse) (64, 65), and has been determined to be significantly misregulated in metastatic prostate cancer based on microarray studies (66). Therefore, we determined if NF-KB signaling was increased in AR- expressing cells. NF-KB p65 activity, as determined by both NF-KB p65 phosphorylation and a reporter assay, were increased in AR-expressing cells (Figure 14 A,B). This is in agreement with previous work that expression of AR in PC3 cells can result in increased NF-KB activity (67). The increase in NF-KB 117 reporter activity was also independent of DHT (Figure 14B). To determine if AR and integrin 06 regulated NF-IcB signaling, AR and integrin 06 were knocked down in AR expressing cells using siRNA. Knock-down of AR or integrin 06 resulted in a decrease in NF-KB p65 phosphorylation and in some cases, a modest decrease in total NF-KB protein levels (Figure 14C,D). TNF0 stimulation of PC3-puro cells was used as a positive control for NF-KB p65 phosphorylation (Figure 14C,D). Thus, NF-KB p65 activity was increased in AR expressing cells in an AR- and integrin 06-dependent manner. To determine if NF-KB p65 (RelA) was regulating BcI-xL expression, AR expressing cells were treated with NF-KB p65 specific siRNA. Knock-down of NF-KB p65 in AR expressing cells resulted in only a partial knock-down of Bcl-xL (Figure 14E), suggesting that another pathway or another NF-KB family member may also be important in regulating Bcl-xL expression in AR expressing cells. Indeed, AR has been reported to directly regulate Bcl-xL expression (51), which could explain why NF-KB p65 knock-down only resulted in partial loss of BcI-xL. Knock-down of NF-IcB did not alter integrin 06 expression (data not shown). Nonetheless, knock-down of NF-KB p65 was sufficient to sensitize AR- expressing cells to LY294002-induced death (Figure 14F). Similar results were seen using the cell permeable small peptide inhibitor SN50, which blocks NF-IcB translocation into the nucleus (data not shown). Thus, the partial knock-down of BcI-xL by NF-KB loss may be sufficient for AR expressing cells to regain dependence on Pl3K signaling. However, NF-KB signaling is known to regulate 118 other key regulators of cell survival, including cFLIP, BFl-1/A1, c-IAP1/2, and XIAP (68), whose loss in expression may also contribute to this phenotype. Classically NF-KB is kept in the cytoplasm bound to a family of inhibitor proteins, called les (inhibitor of KB), where II3’) Ref. Bcl2L1 CGATGGAGGAGGAAG GCACCACCTACATTCA Sun et al., CAAGC AATCC 2008 GAPDH ACCACAGTCCATGCCA TCCACCACCCTG'ITGC Sun et al., TCAC TGTA 2008 ITGA6 GCTGG‘I‘I’ATAATCCTT 'ITGGGCTCAGAACCTT Tapia et al., CAATATCAATTGT GGT'IT 2008 ITGB1 GTGGTTGCTGGAA'ITG TTTTCCCTCATACTTCG Tapia et al., TTCTI'ATT GA‘I'I’GAC 2008 18s CCGCAGCTAGGAATAA CGGTCCAAGAATTI'CA Ottosen et rRNA TGGA CCTC al., 2006 Transfection Assays Cells were plated at B x 105 cells per well in a six-well tissue culture plate on 1% BSA blocked pre-coated 10 pg/mL Iaminin 1 and allowed to grow overnight. Cells were then washed twice and the media was replaced with F-12K media and 10% CSS with no antibiotics. 1.25 pg DNA of pGLB-basic or pGL4.32[Iu02PINF- xB-RE/Hygro], and 0.5 pg thG-TK was incubated with Nanojuice Core Trasfection Reagent and Nanojuice Booster Reagent in Optimem following manufacturer’s directions then added to cells with the following modifications: 4 pl of Core Reagent and 2 pL of Booster Reagent were used per pg of DNA. After 147 48 hours, cells were lysed with Dual-Luciferase Reporter Assay System (Promega) and luminescence measured using EnVision 2104 Multilabel Reader (PERKin Elmer) and Wallac EnVision Manager Software v1.11 following manufacturer’s directions. Firefly luminescence activity was normalized to Renilla luciferase activity. ACKNOWLEDGEMENTS We thank Veronique Schulz, Rich West, Lia Tesfay, and Dr. Brendan Looyenga at the Van Andel Institute (VAI) for technical assistance, and we acknowledge the laboratories of Developmental Cell Biology, Systems Biology, and Cell Structure and Signal Integration at VAI for their constructive suggestions. We also thank Drs. Beatrice S. Knudsen and Peter Nelson (Fred Hutchinson Cancer Research Institute, Seattle, WA), Owen N. Witte (University of California at Los Angeles, Los Angeles, CA), Jeff MacKeigan (VAI), Douglas R. Green (St. Jude Children’s Research Hospital, Memphis, TN), and Anne Cress (University of Arizona, Phoenix, AZ) for generous supply of reagents and Dr. Aaron Putzke (Fred Hutchinson Cancer Research Institute) for AR sequencing primer design. This work was supported by the Department of Defense Prostate Cancer Predoctoral Training Award (W81XWH-08-1-0058) (LL), and the American Cancer Society (RSG-05-245-01-CSM) (J1. and C.K.M). Additional support was also provided by the generous gifts of the Van Andel Institute. 148 REFERENCES 1. 10. 11. American Cancer Society. Cancer Facts and Figures 2009. Atlanta, GA: American Cancer Society; 2009. Liao X, Tang S, Thrasher JB, Griebling TL, Li B. Small-interfering RNA- induced androgen receptor silencing leads to apoptotic cell death in prostate cancer. Mol Cancer Ther 2005 April 1, 2005; 4(4): 505-515. Yang Q, Fung K-M, Day W, Kropp B, Lin H-K. 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Cell Science, 2010. 123: p. 266-276 162 ’ INTRODUCTION Epithelial cells serve several vital functions. For instance, all epithelial cells act as a barrier to protect organs from external environmental assault, as exemplified by the skin. Intestinal epithelial cells are required for the absorption of nutrients, while mammary and prostate epithelial cells are primarily secretory. Proper regulation of epithelial differentiation is crucial for the development and maintenance of barrier and organ function. Differentiation of epithelial cells has been extensively characterized in the epidermis. The basal layer of the epidermis consists of proliferating keratinocytes that adhere to a basement membrane via integrins. Loss of basal cell adhesion through integrin B1 initiates terminal differentiation, resulting in flattening of the cells, expression of differentiation proteins, and subsequent cornification, which ultimately produces several distinct stratified cell layers that make up the epidermis (1, 2). The epithelium of the human prostate consists of two cell layers, a basal layer and a secretory layer. Similar to other stratified epithelium; prostate basal cells are mitotic and adhere to a basement membrane (3-5). Prostate basal cells give rise to terminally differentiated secretory cells (3-5). However, unlike other epithelia, prostate epithelial cell differentiation is regulated by androgen signaling (6-10). The androgen receptor (AR) is a nuclear transcription factor activated in response to the steroid hormone androgen (11). AR is expressed only in the differentiated secretory cells and not in the basal cells (11). It is unclear exactly how androgen regulates epithelial differentiation. However, tissue combination studies from AR null mice suggest that androgen stimulation of AR in the early 163 developing mesenchyme, and not the epithelium, is solely responsible for the induction of epithelial morphogenesis in vivo (12). Androgen also appears to be important for secretory cell survival, in that anti-androgen therapies specifically kill the secretory cells, leaving the basal cells intact (13). Furthermore, restoration of androgens results in regeneration of the secretory cell compartment. However, tissue recombination experiments, as well as studies using conditional knockout mice that lack AR only in prostate epithelium, suggest that AR does not directly regulate epithelial survival (12, 14). Instead, androgen stimulation of the AR-positive stromal cells of the prostate may induce secreted factors that regulate secretory cell survival. Keratinocyte growth factor (KGF) and FGF 10 are two factors secreted by the stromal cells, though not in an androgen-dependent manner (12, 15-17). KGF and FGF10 are both involved in murine prostate organogenesis and can induce differentiation of isolated prostate epithelial cells (15, 16, 18-22). In some cases, KGF can substitute for androgens and it is likely that KGF and AR signaling pathways interact (23). KGF has also been reported to promote differentiation and survival of the epithelium of the skin, lung, and eye (24-26). KGF acts specifically on epithelial cells and has been reported to activate p38 MAPK signaling (21). Clarification of the roles of androgen and KGF in prostate epithelial differentiation and survival has been hampered by our inability to culture normal differentiated AR expressing secretory cells in vitro. Prostate epithelial cells (PECs) cultured from normal human prostate tissue consist primarily of AR- negative basal cells and their transient amplifying derivatives. Previous studies 164 in our lab have demonstrated that survival of cultured PECs is specifically mediated through 0381 integrin-dependent adhesion (27). Similarly, basal keratinocytes are dependent on 0381 integrin for their survival (28). During keratinocyte differentiation, basal cells lose integrin expression as well as adhesion to matrix as they are extruded to the upper layers of the skin (29). In suprabasal keratinocytes, as well as in other epithelia, cell-cell adhesion structures such as E-cadherin appear to promote survival through phosphoinositide 3-kinase (PIBK) signaling, and when PIBK signaling is lost these cells die (30-32). Whether the same survival mechanisms are operative in differentiated secretory prostate epithelial cells is unknown, and the role of KGF or androgen in prostate epithelial cell survival remains unresolved. In this study, confluent cultured primary prostate basal epithelial cells were induced to differentiate following treatment with KGF and androgen. After two weeks, differentiated AR-expressing secretory cells appeared as a secondary cell layer above the basal cells. This model was used to identify the signaling pathways important for prostate secretory cell survival. This new model will serve as a valuable tool for understanding the biology of prostate secretory epithelial cells, a cell population previously not available for extensive analysis. RESULTS Differentiation of Confluent PECs by KGF and DH T Previous studies have demonstrated that KGF may be an important epithelium differentiation factor in many tissues, including prostate epithelium 165 (15, 16, 19, 21, 33). Androgen, acting via the androgen receptor, also plays an important role in prostate epithelial cell differentiation (6-10). To determine if the combination of KGF and androgen is sufficient to induce differentiation of prostate cells grown in culture, human primary basal prostate epithelial cells (PECs) grown to confluency in monolayer cell cultures were treated with 10 ng/ml KGF and 5-10 nM androgen (DHT). Culturing the cells for 10-15 days with KGF/DHT resulted in the formation of stratified cell patches consisting of at least two cell layers, resembling the bilayer of basal and secretory cells observed in the prostate epithelium in vivo (Figure 17A,B,C). To determine if the stratified cells expressed differentiation markers specific to prostate secretory cells, expression of AR and the AR-target protein prostate-specific antigen (PSA) were examined by fluorescence confocal microscopy. Cells in a higher z-plane than the bottom cells, stained positive for AR and PSA (Figure 178). AR expression was both nuclear and cytoplasmic, whereas the secreted protein PSA had the expected cytoplasmic localization (Figure 17B). While AR expression was uniform throughout the top cells, PSA expression was often concentrated at the upper membrane of the top most cells, consistent with that of a secreted protein (not shown). Neither AR nor PSA was found in the bottom cells (Figure 17B). Additionally, the AR-regulated proteins ka3.1 and TMPRSS2 (34-37) were expressed in top cells and not in bottom cells (Figure 17C). To determine the extent to which androgen stimulation contributes to PEC differentiation, PECs were treated for 10-14 days with KGF in the presence or 166 absence of DHT, and the expression of AR, AR target proteins, and differentiation cell markers was monitored. PSA, ka3.1, and TMPRSSZ were only expressed when DHT was present (Figure 170, PSA and TMPRSSZ not shown). lntriguingly, cytokeratin markers, K18 and K19, were also expressed only in the presence of androgen (Figure 170, K18 data not shown). Furthermore, there was a dramatic increase in AR expression itself when DHT was present. KGF, in the absence of DHT, was sufficient to induce formation of stratified cells, with maximal formation occurring between ten to fifteen days. PECs treated with KGF in the presence of KGF-blocking antibody did not stratify. Confluency of the cultures was essential. Subconfluent cells treated with KGF and DHT did not form stratified clusters. KGF-induced stratification occurred equally efficiently, with or without the supplementary bovine pituitary extract (BPE) and EGF in the culture medium. Occasionally, a few small stratified clusters appeared in BPE-containing medium without KGF treatment, suggesting the presence of low levels of KGF and/or an additional unknown factor(s) in BPE that can promote differentiation at a low efficiency. KGF-blocking antibodies prevented the appearance of these occasional clusters. The optimal concentration of KGF was 10 ng/ml. Lower doses (1-5 ng/ml) resulted in fewer clusters and higher doses (20-50 ng/ml) did not generate more clusters. DHT alone was not sufficient to induce stratification. DHT plus KGF treatment dramatically increased the number of top cells seen after fifteen days. DHT was 167 ‘I FIGURE 17. AR and AR-dependent proteins are present in the differentiated cultures. Confluent primary prostate epithelial cells (PECs) were induced to differentiate with 10 ng/ml KGF and 5nM DHT for 10-14 days. Scale bars: 100 pm. A) DIC image of a differentiated culture shows an upper layer of cells (outlined with dashed white line) on top of a confluent bottom cell layer. B) 14-day differentiated culture was immunostained for AR (green) and PSA (red). Nuclei (blue) were visualized by Hoechst 33258 staining. (Left panel) A z-section image was compiled from 30 confocal x-y sections representing a thickness of 38.0pm. Horizontal lines demarcate top and bottom cell layers. (Right panels) Confocal images of top cells in 14-day differentiated cultures. Scale bar: 50.0 pm. C) Differentiated PEC cultures were immunostained for ka3.1 and TMPRSSZ (green) and imaged by confocal microscopy. Nuclei were stained with Hoechst 33258 (blue). Representative top and bottom cells and z-plane images are shown (Z). Scale bar: 100 pm. D) PECs were induced to differentiate for 14 days with KGF in the presence or absence of 10 nM DHT. Cells were immunostained with AR, kaB.1, and K19 and imaged by epifluorescence microscopy. 168 FIGURE 17 B. _- C. ka3.1 TMPRSSZ Z-Plane DHT+ p N Ibottom top DHT- 169 required for expression of androgen-dependent markers in the top cells. FGF 10, a functionally related FGF family member shown to be important for prostate development in vivo (22, 38), could also induce PEC differentiation in the presence of DHT. Differentiation was reproducibly observed in cells derived from two different patients at three different passage numbers (passages 2, B, and 4). It was observed however, that once cells reached passage 5, the efficiency of differentiation was dramatically reduced. Furthermore, we were able to induce differentiation in an immortalized cell line derived from a third patient. We observed that these more proliferative immortalized cultures took a few days longer to reach maximal differentiation. Stratified Cells Express Additional Differentiation Markers Markers specific to basal and differentiated epithelial cells populations were examined in the stratified cultures. The basal markers Bcl-2, K5, and K14 (39, 40) were expressed predominantly in the bottom cells; occasionally a few K5- and K14- positive cells were seen in the top cells (Figure 18A,B, K14 not shown). Basal marker p63 (41, 42) was associated only with bottom cells (Figure 2A). EGFR, which is predominately expressed in basal cells (43), was associated primarily with bottom cells (not shown). Epithelial cell markers K19 and PMSA were expressed only in the top cells and not in the bottom cells (Figure 18B,C). K18, as well as the cell cycle inhibitor p27 (Kip1) (44-47), was expressed predominately in the top cells (Figure 18C). Z-plane analysis of cells co-stained for Bcl-2 and p27 (Kip1) demonstrate the co-distribution of a basal cell 170 FIGURE 18. Differentiation-specific epithelial markers present in the top cells of differentiated cultures. 10- to 14-day differentiated cultures were immunostained for A) Bcl-2, p63 (green), B) K5 (green), PSMA (red), C) K18, K19 (red), and p27/Kip1 (green) expression and images were captured by A,C) confocal microscopy or B) epifluorescence. Nuclei were stained with Hoechst 33258 (blue). Representative top and bottom cells are shown. Representative z-section images (2) were compiled from 10-15 confocal x-y sections representing a thickness of 17.04 (+/- 3.27) pm. Horizonal lines demarcate top and bottom cells. Scale bars: 100.0 pm. D) 11-day differentiated cultures were immunostained for basal marker Bcl-2 (red) and differentiated marker p27/Kip1 (green). Nuclei were stained with Hoechst 33258 (blue). Z-section image was compiled from 20 confocal x-y sections representing a thickness of 28.64 pm. Scale bar: 50 pm. 171 FIGURE 18 p63 PSMA = Red Nuclei = Blue K5 = Green Top Bottom p27/Kip1 p27IKip1 = Green Nuclei = Blue Bel-2 = Red 172 marker and a differentiation marker, respectively, in the stratified cultures (Figure 18D). Differentiation Induces Integrin Loss Consistent with previous observations of differentiating epithelium in vitro and in vivo (1, 21, 48, 49), epifluorescence and confocal imaging revealed that the subpopulation of the cells undergoing differentiation lost expression of many integrins, including 02, (13, d6, 81, and 84 (Figure 19A,B). Basal cells also expressed dv, but not [33 or 85 integrin subunits. None of these integrins were present in the differentiated cells (not shown). Cultured PECs secrete and organize a Iaminin 5 (LM5)-rich matrix (50); the differentiating cell population that lost integrin expression also no longer produced LM5 (Figure 19A,B). Although it appears by confocal imaging that the cells directly below the top cells do not express integrin or LM5, it is possible that there is incomplete antibody penetrance into the lower cells. To address this, a time course study was performed. We observed a decrease in LM5 expression as early as three days after KGF and DHT treatment and a complete loss after eight days. At eight days decreased 81 integrin expression was observed in LM5-negative cells prior to formation of the second cell layer (Figure 20A,B). Therefore, cells directly underneath the top layer also lose LM5 and integrin expression. LM5 loss may be the trigger that initiates differentiation. 173 Phase Contrast ITGB1 Z—_—-__ FIGURE 19. Prostate epithelial differentiation is accompanied by loss of integrin expression. A) Integrin I31 (ITGB1, green) and Iaminin 5 (LM5, red) expression in 10 day differentiated cultures were monitored by DIC (left panel) and epifluorescence microscopy following immunostaining. B) 14-day differentiated cultures were immunostained to detect expression (green) of integrins (12 (ITGA2), d3 (ITGAB), c6 (ITGA6), I31 (ITGB1), B4 (ITGB4), and Iaminin 5 (LM5) and imaged by confocal microscopy. Nuclei (blue) were visualized by Hoescht 33258 staining. Representative confocal x-y sections of the top and bottom cells are shown. The area located directly beneath the top layer of differentiated cells is indicated with dashed white lines in the bottom image. Representative z-section images (2) were compiled from 10-15 confocal x-y sections representing a thickness of 17.04 (+/- 3.27) pm. Horizontal lines demarcate top and bottom cells. 174 Hoescht _ . overlay Day 4 ' a ‘-.._1 . ' ..‘ [V I3, ~ -' , ‘ Day 7 .9 , ,I '\ . I, \ I . " \ 0 - r. \ ’ > ‘ A . I .>‘ - ,. ‘ \ I. , . I \ . . .3 . . _. I I ‘ , ‘ x I ’ . \ r . , x I ~. .r I . ~ 'gi- .'.‘.~ ,f ~‘u L " Day 11 oveflay A Day 4 O ‘ ‘ . . r, .y, Day 7 I a Day 11 FIGURE 20. Prostate epithelial differentiation is accompanied by loss of Iaminin 5 expression. Confluent PECs were induced to differentiate in the presence of KGF and DHT for 4, 7 or 10 days. Cells were immunostained (red) for A) Iaminin 5 (LM5-y2) or B) [31 integrin (ITGB1) and Hoechst 33258 (blue) and visualized by epifluorescent microscopy. Areas of decreased LM5 and B1 integrin expression are outlined with dashed white lines. 175 Differentiated Cells Respond to Androgen AR expression could be detected by immunoblotting of cell lysates from whole cultures treated with KGF and DHT (Figure 21A). Expression of the androgen- dependent secreted proteins, KLK2 and PSA, was monitored in differentiated cultures by RT-PCR. KLK2 and PSA mRNAs were present only when DHT was present in the culture (Figure 218). Furthermore, secreted PSA, up to 0.8ng/ml, could be detected by ELISA (Figure 21C). PSA secretion required androgen and increased with increasing DHT concentration. The expression and secretion of an androgen-regulated protein in an androgen- dependent manner indicates the presence of differentiated prostate secretory cells in the culture, and that AR is functional and regulates expression of differentiation markers. Overall, this KGF and DHT-induced in vitro differentiation model recapitulates many aspects of in vivo differentiation as assessed by the specific markers (Fig 21D). In addition to the induction of markers common to most differentiating epithelial cells, the presence of DHT markedly stimulates the expression of markers unique to prostate secretory epithelial cells. Hereafter when referring to this model, the AR-expressing top cells will be referred to as secretory-like cells and the AR-negative bottom cells as basal cells. Isolation of Secretory-like Cells Treatment of differentiated cultures with dissociation buffer preferentially dislodges the secretory-like cells. FACS analysis indicates that 96.6% (+/— 0.8%) 176 A. PEC: LN D, EpIthoIIaI Markers + - - KID I In Vlvo In Vltro , Marker asal lef. Bottom T AR ' I— i 3°” ‘. ‘5 I EGFR __ ‘ Tub -I-—I K14 h. —__ K5 ‘ ¥ 8. PEC: LN P53 ‘_ ‘. 927 4 AI ITGA2 ‘ “ "6A3 - ‘ "GAB ‘ - ITGB1 ‘ I ITGB4 I - 1 . LNG I I C. 0,8 - Androgen-Regulated Markers In Vlvo InVlIro— < 0.6 - 2 AR I I E 0.4 - PSA - - 3 o 2 . ka3.1 I - : TMPRSSZ - A 0 . PMSA - I .0 2 . 0 2.5 5 10 25 K19 4“ I ' Conc. DI-IT (nM) K13 A A FIGURE 21. Differentiated cells respond to androgen. A) lmmunoblot for AR expression in cultures of PECs treated with or without KGF and DHT (KID) for 16 days. LNCaP cells (LN) were used as a positive control for AR expression. Total levels of protein in the lysates were monitored by immunoblotting with anti- tubulin. 8) Levels of KLK2 and PSA mRNA isolated from 14-day differentiated (KID) cultures were analyzed by PCR and compared to LNCaP (LN) cells. GAPDH served as a control. C) Secreted levels of PSA from 14-day differentiated cultures treated with KGF and increasing amounts of DHT were determined by ELISA. D) Summary of epithelial and androgen-dependent markers observed and their relative expression in the lower (Bottom) and upper (Top) cells. Expression observed in vitro is compared to that reported in vivo. 177 of the isolated dislodged population is negative for cell surface (:6 integrin, while 97.19% (+/- 1.70%) of the cells not dislodged are positive for 06 integrin (Figure 22A). Further FACS sorting based on surface staining of (:6 integrin and TMPRSSZ revealed that on average 87.92% (+/- 3.71%) of the d6-integrin- negative cells were positive for TMPRSS2 A representative example is provided in Figure 22B. Immunoblotting of separated cells indicated that some remaining basal cells expressed AR as well as full-length TMPRSSZ protein; however, only the secretory-like cells expressed the cleaved and activated form of TMPRSSZ (Figure 22C) (51). Conversely, only the basal cells expressed Bcl-2 and EGFR, while K5 was predominately found in the basal cells (Figure 220). Secretory Cell Survival is Dependent on PI3K and E-cadherin, but not KGF or Androgen In previous studies we demonstrated that integrin-mediated activation of EGFR and downstream signaling to ERK, but not PIBK signaling, is required for the survival of basal PECs (27). However, the differentiated secretory-like PECs have lost integrin expression, no longer adhere to the LM5 matrix, and have significantly lower levels of EGFR, suggesting that other survival pathways must be important for secretory cell survival. It has been suggested that secretory cell survival may be dependent on stromal derived growth factors, including KGF (52). One possibility is that the KGF used to induce differentiation, may also be necessary for survival. To test this, the KGF receptor FGFR2lIlb (53) mRNA levels were analyzed in the isolated secretory-like cells and basal cells by RT- 178 A. Bottom ng I ITGA6+ ITGA6-I- E eflrh 4+ 0.8 E 2.81% -/+ 1.70 3 . 8 o I o .. k Fluorescence Fluorescence Key: — Rat lgG —- ITGA6 Top c B 1 AR - FL TMP - :: SP TMP - "—7: TubuIIn - - _- FIGURE 22. Isolation of secretory-like cells. A) Following treatment of 14-day cultures with dissociation buffer the separated upper (Top) and lower cells (Bottom) were subjected to FACS to measure cell surface a6-integrin expression (gray line) versus control antibody (black line). Dead cells were excluded using Pl staining. Values are percentage d6-integrin-positive cells. B) Cells isolated and sorted for G6 integrin as in A were further sorted based on TMPRSSZ (TMP) surface expression. Values are percentage of positive cells in each quadrant. Data is from one typical experiment. C,D) Bottom (B) and top (T) cells, obtained after treatment with dissociation buffer, were analyzed by immunoblotting for C) AR, TMPRSSZ, D) Bcl-2, K5, and EGFR expression. Tubulin (Tub) immunoblotting served as a loading control. 179 PCR. Only the basal cells expressed FGFR2lllb mRNA (Figure 23A). Furthermore, removal of KGF after 15 days of differentiation did not induce cell death (not shown). Thus it is unlikely that KGF is regulating cell survival in the secretory-like cells. Dissociated secretory-like cells and the remaining basal cells were screened for ERK and Akt activation by immunoblotting. Active ERK was present only in the basal cells, but not in the secretory-like cells (Figure 23B). Activated Akt was present in both types of cells (Figure 23C). Thus, ERK signaling probably does not regulate survival in differentiated cells, whereas the PIBK pathway could. Since the differentiated cells remain adherent to the bottom basal cells, we also investigated whether there is an increase in expression of the cell-cell adhesion molecule E-cadherin in the secretory-like cells. Compared to the basal cells, E-cadherin levels were elevated in the secretory cell population that also does not express c681 integrin (Figure 23D). E-cadherin can lead to activation of PIBK signaling in skin and colonic epithelium as well as in some tumor cell lines (30, 54, 55). Blocking antibodies to E-cadherin suppressed Akt activity in both the secretory-like (Figure 23E) and the basal cells (not shown). The relative importance of the different signaling pathways on secretory- like cell survival was investigated. Fourteen-day KGF and DHT-differentiated cultures were placed in KGF- and DHT-free basal medium without any pituitary extract or EGF supplement for 72 hours to reduce any signaling induced by the growth medium (Figure 24A). Visually, the starved cell cultures appeared viable, 180 A. B T B. B T Fem-_ ”ME. I GAPDH - Efk‘I -I — -I c. e r o. e r P-Akt-I——-J Ecad-I- _I Akt-I—-I : ITGA6 - III—- I Tub - I-q Tub - I——I E. lgG Ecad P-Akt - I u- I Akt-[2.] Tub-I—-I FIGURE 23: Signaling pathways in secretory-like cells. Following treatment of 14-day cultures with dissociation buffer, mRNA or protein was isolated from the lower (B) and upper cells (T). A) Levels of FGFR2lIlb mRNA (FGFR2) were assessed by PCR. GAPDH served as a control. B) Levels of ERK activation (P- ERK) in the lower (B) and upper cells (T) were monitored by immunoblotting of cell lysates with phosphospecific ERK antibodies. Total levels of ERK and tubulin in the lysates were measured by immunoblotting. C) Levels of Akt activation (P-Akt) in the lower (B) and upper cells (T) were monitored by immunoblotting of cell lysates with phosphospecific Akt antibodies. Total levels of Akt and tubulin in the lysates were measured by immunoblotting. D) Levels of E-cadherin (Ecad) and d6 integrin (ITGA6) in the lower (B) and upper cells (T) were analyzed by immunoblotting. Tubulin immunoblots served as loading controls. E) 14-day cultures were treated with control lgG or E-cadherin-blocking antibody (Ecad) for three hours. Levels of Akt activation (P-Akt) in the isolated upper cells were monitored by immunoblotting of cell lysates with phosphospecific Akt antibodies. Total levels of Akt and tubulin in the lysates were measured by immunoblotting. 181 and the upper secretory-like cell layer remained intact (data not shown). Then the starved differentiated cultures were treated with specific inhibitors in the presence or absence of freshly added DHT or KGF and analyzed over a 72 hour time course. Cell death was measured in the upper secretory-like cell layer by immunostaining for active caspase 3/7, TUNEL staining, or propidium iodide (PI) uptake. Staining was quantified as described in Materials and Methods. Inhibition of Pl3K signaling with LY294002 resulted in maximal secretory-like cell death at 72 hours, where 60% of the cells stained positive for PI (Fig 248). Furthermore, inhibition of PI3K, but not EGFR, induced a 7.0- to 7.5-fold increase in secretory cell caspase 3 activity (Fig 24C), and a 5.5- to 5.7-fold increase in TUNEL staining (Figure 24D,E). Maximal Annexin V staining was observed 66 hours after LY294002 treatment (not shown). Secretory-like cell survival was not dependent on DHT or KGF, and addition of DHT or KGF was unable to promote cell survival in the absence of PI3K signaling (Figure 24B-D). Although KGF should not be present in the media and prostate epithelial cells have been reported not to produce KGF, KGF-blocking antibodies were used to prevent any endogenous or remaining KGF from promoting cell survival. KGF-blocking antibodies had no effect on cell survival (data not shown). KGF has been reported to activate p38, and Jnk can promote survival during stress (21, 56, 57). Inhibiting p38 with 88202190, Jnk with 420119, or ERK with PD98059 did not result in cell death, suggesting these pathways are not critical for secretory cell survival (Figure 24F). The lack of effect of the inhibitors on cell survival was not 182 FIGURE 24. Secretory-like cells are dependent on PI3K and E-cadherin, but not androgen or KGF, for survival. A) PECs were treated with KGF and DHT for 14 days (Differentiation), then starved of growth factors and DHT for 3 days (Starv), and then treated with pharmacological inhibitors (Drugs) for 1-3 days. In some cases DHT or KGF were also added back with the inhibitors. B) Differentiated cultures were treated with vehicle (DMSO) or PI3K inhibitor LY492004 (LY) in the presence or absence of DHT (D) for 24, 48 or 72 hours. Cell viability in the top cells was measured by quantifying the . number of cells with high PI staining and expressed as percentage Pl-positive cells. C, D, E) Differentiated cultures were treated with vehicle (DMSO), EGFR inhibitor PD168393 (PD1), or PI3K inhibitor LY492004 (LY) in the presence or absence of DHT (D) or KGF (K). After 72 hours cell viability in the top cells was assessed using C) cleaved caspase-3 or D) TUNEL staining. Total DNA was stained with Pl. Six fields per experiment and condition were examined and positive pixels counted using the software program Imagine as outlined in Material and Methods. TUNEL- or cleaved caspase-3-positive pixels were normalized to the total number of stained DNA pixels in the region of interest and expressed as relative intensity of caspase- 3 or TUNEL staining. Error bars are standard deviation. n=3. E) Cell viability in the upper cells was accessed using TUNEL (green) staining and confocal microscopy. Nuclei were stained with propidium iodide (PI, colon'zed blue). F) 14 day differentiated cultures starved of growth factors for 3 days were treated with vehicle (DMSO), MEK1/2 inhibitor PD98059 (P09), p38 inhibitor 88202190 (SB) or Jnk inhibitor 420119 in the presence of KGF and the presence or absence of DHT. Cell viability was measured by TUNEL staining 3 days later. Error bars are standard deviation. n=3. G) Differentiated cultures were treated with non-specific mouse lgG (lgG) or with E-cadherin blocking antibody (Ecad Ab; lot 2) in the presence or absence of DHT (D) for 24 or 48 hours. Cell viability was measured by PI staining. Error bars are standard deviation. n=3. H) Cell viability of differentiated cultures treated with non-specific mouse lgG (lgG) or with E-cadherin blocking antibody (Ecad Ab; lot 1) in the presence or absence of DHT (D) or KGF (K) for 72 hours was measured by TUNEL staining. Error bars are standard deviation. n=3. 183 FIGURE 24 A. B. 100 g 80 Experimental Time Line: .2 50 § 40 l I I I n- 20 I l l I E 0 Differentlatlon Stunt. Drugs ‘2 14d 3d 1-3d ° (3.5.100 - D' > . '2 80 8 60 1 a. 40 - 8 2o , a o - 8 0 KEY: I I96 I Ecad Ab 184 1oo . so J 60 - 4o - 20 - % TUNEL Posltivity O l NT scram slAR STR FIGURE 25: AR is not required for secretory-like cell survival. PECs were treated with KGF and DHT for 14 days and then transfected with scrambled siRNA (scram) or AR-specific siRNA (siAR) to block AR expression. A) Cells were immunostained with AR, ka3.1, and K19 (green) and imaged by epifluorescence microscopy. Nuclei were stained with Hoechst 33258 (blue). B) Differentiated cultures left untreated (NT) or treated with scrambled siRNA (scram), AR siRNA (siAR), or staurosporine (STR) were measured for cell viability by TUNEL staining. 185 due to a failure to inhibit signaling, as the concentrations of drugs used here did effectively block signaling to their specific targets in basal cells. Inhibition of cell-cell adhesion with one application of E-cadherin blocking antibody resulted in maximal cell death at 48 hours with over 80% of the cells staining positive for PI (Figure 24G). By 66 hours, no secretory-like cells remained in the cultures. A second application of E-cadherin antibody resulted in a 7- to 8-fold increase TUNEL staining 72 hours after treatment (Figure 24H). The presence of DHT or KGF could not protect cells from death due to loss of E- cadherin function. No cell death was observed in the lower basal cells. Furthermore, blocking E-cadherin lead to a decrease in Akt activation (see Figure 23E), indicating that cell-cell adhesion mediated by E-cadherin promotes secretory-like cell survival through PIBK signaling. Although DHT was not important for survival of the differentiated secretory-like cells, it is theoretically possible that AR, acting via an androgen- independent mechanism might still be important for cell survival. To address this, 14-day KGF and DHT-differentiated cultures were transfected with an AR- specific siRNA pool or a scrambled siRNA sequence. Confocal imaging of the transfected cells 72 hours later demonstrated the absence of AR expression in the upper cells (Figure 25A). Absence of AR expression also resulted in loss of androgen-dependent cell markers such as ka3.1 and K19 (Figure 25A). Cell viability of the AR siRNA-treated cells was assessed by TUNEL staining. Loss of AR had no effect on secretory-like cell viability (Figure 258). Thus, AR and 186 androgen signaling are not required to maintain the viability of differentiated secretory-like cells derived from our in vitro culture system. DISCUSSION By treating cultured primary prostate basal epithelial cells with androgen and KGF, we have established an in vitro differentiation model of the prostate epithelium. The differentiated cells in our culture system possess the important features of terminally differentiated secretory prostate epithelial cells in vivo: they do not proliferate, they adhere to a basal cell layer and not to the basement membrane, they express AR protein, and they respond to DHT by inducing AR- dependent genes. Specifically, the cells express androgen-sensitive proteins, such as KLK2, PSA, ka3.1, PMSA, and TMPRSSZ. In addition, cleaved TMPRSS2 is present in the upper, but not the lower cells and PSA is secreted into the culture medium. Furthermore, cytokeratin K18 and K19 expression was found to be dependent on androgen. K18 expression has previously been reported to be regulated by androgen (8, 9), and K19 has been suggested to be responsive to estrogen (58); however, both K18 and K19 promoters lack classical androgen response elements, making the mechanism of regulation unclear. Further evidence for terminal differentiation is that the cells did not revert to basal cells when isolated and re-plated, and they failed to reattach, likely due to continued loss of integrin and/or matrix expression. Furthermore, after 21-25 days in culture the upper cells sloughed off and a few activated caspase 3 positive cells were seen in the aging cultures (data not shown), similar to what is 187 observed in vivo. Oddly, no more differentiated cells reappeared. Only about 20% of the cells appeared to be capable of undergoing differentiation, suggesting that the differentiated cells are derived from a distinct suprpulation of basal cells. The lack of continued differentiation after 25 days may indicate depletion of these special cells and a lack of ability to renew. The population of differentiation-competent cells is not likely to be stem cells, since 20% of the cells are capable of undergoing differentiation. However, we can’t rule out the possibility that these cells arose from some stem cell-like progenitor within the culture. Further analysis would be required to determine if the progenitors are analogous to the ka3.1 positive luminal stem cell recently described (59). However, whatever the progenitor, it apparently can’t renew in the context of our culture conditions. Although many aspects of the differentiated cells recapitulate what is observed in vivo, there still remain some differences. For instance, the distribution of AR demonstrates a significant amount of cytoplasmic expression in the in vitro culture system, while in vivo AR is primarily nuclear. Another difference is the absence of columnar cells. In addition, a few K5- and/or K14- positive cells were sometimes seen in the upper layer, which has also been reported in another differentiation model (60). Hence, we cannot unequivocally say whether our secretory-like cells represent completely terminally differentiated prostate cells and there are still some distinctive morphological differences between our cultures and what is seen in the prostate gland in vivo. 188 Other studies have reported on prostate epithelial differentiation in vitro. While these studies were informative, they were limited since AR and AR- regulated proteins were not expressed (48, 61-65). A few studies have reported seeing stratified layering similar to ours after treating prostate epithelial cells in vitro with retinoic acid, FGF, and/or insulin (44, 48, 60, 66); however, in these models the top layer of cells either failed to express AR or still expressed basal markers. In our model, the top secretory-like cells expressed AR and lost basal marker expression. In one case, gland-like buds and extensions were observed to form from confluent cell cultures, reminiscent of acini structures in overall shape but without lumens (60). We have also observed cases where cells appear to form mounds. By confocal imaging, some of them appear to have formed a hollow mound (data not shown). A recent study demonstrated that co- treatment of prostate basal cells with the monoamine oxidase A inhibitor clorgyline, 1,25- dihydroxyvitamin D3, all-trans retinoic acid, and TGF-[31 induced AR expression and loss of basal marker K14 (67), suggesting that there may be alternative mechanisms to inducing prostate epithelial cell differentiation. In contrast to other published systems, we have demonstrated that our model can be utilized for biochemical and genetic manipulation. It is amenable to treatment with pharmacological inhibitors or siRNA to study signaling and biological pathways. Furthermore, exploitation of differential cell surface markers and adhesion properties can be used to separate basal from secretory-like cells to separately analyze RNA and protein expression. 189 1h. It is unknown if AR represses integrin expression or if loss of integrin expression must precede expression of AR. Unpublished data from our laboratory and others demonstrates that re-expression of AR in prostate cancer cell lines results in decreased integrin expression (68, 69). However, in our model we observed that not all integrin-negative cells were AR positive, suggesting that integrin loss may precede AR expression. Furthermore, LM5 matrix loss preceded integrin loss, which preceded stratification and robust AR expression in our time course studies. Heer et al. have demonstrated that blocking integrin B1 is sufficient to induce partial differentiation; however, cells do not reach terminal differentiation since the cells do not express AR-regulated genes (21). This suggests that loss of adhesion can initiate early differentiation and may even be required, but that integrin loss alone is not sufficient for terminal differentiation. In contrast, unbound integrin [31 is sufficient to initiate terminal differentiation in keratinocytes (1, 29). On the other hand, in mammary epithelium loss of integrin I31 suppresses differentiation (70). Interestingly, in most of the reported prostate differentiation models (including ours), confluent cultures were necessary for stratification. In addition, previous studies suggest that cell cycle inhibition is a prerequisite for expression of secretory cell markers K18, K19, and AR (48, 62, 65, 71). We similarly saw a loss in cell proliferation in the differentiating cell population (data not shown). This led us to develop the following model for prostate differentiation (Figure 26). Basal cells are proliferative and a subset begins to undergo growth arrest once the cells are confluent. Treatment with KGF causes a select population of cells, 190 Trans-amplifylng O Secretory Precursor - E-cadherln 0 Mature Basal Q Mature Secretory I Integrins - Matrlx FIGURE 26: Model of Differentiation. A) Confluent primary prostate basal cells secret and adhere to a matrix rich in LM5 via integrins, which physically separates the epithelial cells from the stromal cells. B) After treatment with KGF, a sub-population of transient amplifying cells loses expression of LM5 and subsequently integrins, resulting in loss of adhesion. Concurrently, there is increased cell-cell adhesion via E—cadherin. C) Secretory-like precursor cells arise in concert with androgen treatment, which induces their differentiation into mature secretory cells. Transient amplifying cells at the edge continue to proliferate to fill in the space generated by detachment of cells and movement into the top layer. After 10-14 days, cells become stratified as more transient amplifying cells are committed to terminal differentiation. 191 perhaps those that express higher levels of the KGF receptor FGFRZIIIb (53), to lose LM5 and then integrin expression, causing the cells to detach. Integrin loss and detachment may then trigger low AR expression. AR expression was not detectable by immunostaining in cultures treated with only KGF, where integrin ' expression was lost; however, some AR expression was detectable in the basal cells from the differentiated cultures by immunoblotting. The presence of androgen in the culture appears to be necessary to allow the integrin-deficient cells to express AR at a higher level, which then turns on AR-dependent differentiation-specific genes. Work by Heer et al. suggests that AR may be expressed at low levels in primary prostate epithelial cells and is rapidly degraded by the proteosome (8); hence androgen treatment may stabilize and/or help drive production of AR protein. In fact AR mRNA has been detected in some cultured prostate epithelial cells (71). However, in our studies and those of others, androgen alone is poorly effective in inducing AR expression (71). Thus, additional events are required to induce stable AR expression even in the presence of androgen. Reduced cell proliferation caused by strong growth suppression or loss of cell adhesion, which is also growth suppressive, may be necessary. Significant increases in AR expression can be detected in isolated suspended cells in the presence of androgen (8), thus supporting cell detachment as a potential mechanism required for stabilizing AR. Previous work from our laboratory has demonstrated that integrin- mediated survival of primary prostate basal cells requires integrin-induced EGFR 192 signaling to ERK, but not PI3K signaling (27). In this study we have expanded our analysis of survival mechanisms to secretory-like prostate epithelial cells and demonstrated that secretory-like cells depend on a non-integrin-dependent mechanism for cell survival that involves cell-cell interactions through E-cadherin. Interestingly, there is switch from ERK-dependent survival in the basal cells to PI3K-dependent survival in the secretory-like cells. In the secretory-like cells EGFR levels dropped dramatically and EGFR-dependent signaling to PI3K was not required for survival (blocking EGFR had no effect on secretory cell survival). Interestingly, in prostate cancer, there appears to be a strong dependence on PI3K signaling for survival, as these cells tend to acquire mutations in Pten, a negative regulator of Pl3K signaling (27, 72-74). This suggests that prostate cancer may arise from a more differentiated cell that has already acquired dependence on PI3K for its survival. In our studies, secretory cell survival was not dependent on the presence of androgen, and knock-down of AR with siRNA in differentiated cells did not induce their death. The lack of dependence on androgen or AR for secretory cell survival in our human culture system is in agreement with genetic and tissue recombination studies in mice. Conditional knock-out of AR in mature mouse prostates results in decreased numbers of secretory cells without inducing cell death, suggesting that AR functions to increase secretory cell numbers by promoting differentiation rather than cell survival in mature glands (14). Tissue recombination experiments using mesenchyme and epithelium from AR-negative or wild-type mice demonstrate that AR expression in the epithelium is not 193 required for early prostate development, indirectly ruling out a role for AR in epithelial cell survival in newly formed glands (12). Thus, in both models, as well as ours, androgen is responsible for the synthesis of secretory proteins and the secretory function of the prostate. If androgen and AR do not act cell autonomously to control epithelial cell survival, then why do only the AR-expressing epithelial cells die upon castration- induced androgen deprivation (75, 76)? One possibility is that AR signaling in the stromal cells promotes survival by paracrine factors that act on the epithelial cells (77). In our model the paracrine function of KGF, known to be expressed by stromal cells in vivo, was required for differentiation; however, it was dispensable for cell survival in committed differentiated cells. Thus, the nature of the paracrine survival factor(s) remains undetermined. In our in vitro model, survival was highly dependent on E-cadherin-based cell-cell adhesion and signaling to Pl3K. Whether paracrine factors in vivo are responsible for maintaining survival via E-cadherin or whether they act on other pathways remains to be determined. Our study supports a simpler concept that the role of stromal-derived paracrine factors is to act primarily on the stem and/or basal cells, whose proliferation and regenerative capacity is driven by these factors. As terminally differentiated cells are sloughed into lumen, basal cells are triggered to proliferate and differentiate to replace the lost cells. Under androgen-ablative conditions, the loss of paracrine factors in the stroma prevents stem and/or basal cell renewal and the terminally differentiated cells eventually slough off and are Ulflrfl rfl rfllffi UQHDJUDIII Ill rl] rfl Hi 11% “(LB [1815230 II] III 194 differentiation, and subsequent restoration of secretory cells. This model would preclude the need for stromal factors acting directly on the secretory cells. An alternative model to explain castration-induced loss of prostate secretory cells involves the observation that castration reduces blood flow and microvasculature collapse in the gland, inducing a state of hypoxia (78). It would appear that secretory cells are much more sensitive to such stress than the basal or stromal cells. This may be related to a lack of extracellular matrix support that provides additional survival signaling cues to the basal and stromal cells. Alternatively, hypoxia may affect the production of the paracrine factors required for maintenance of epithelial differentiation or survival. In summary, we have established an in vitro differentiation model of human prostate epithelium composed of stratified cells that recapitulates many in vivo characteristics of basal and secretory cells, including AR-dependent differentiation and function. This model can be treated with pharmacological inhibitors and siRNA to study biochemical and genetic effects and the differentiated secretory-like cells can be isolated for further analysis. We have further established that while KGF, AR, and androgen are important for initiating the differentiation process and AR is important to maintain the androgen- dependent phenotype of secretory-like cells, these factors are not required for survival of the committed differentiated cells. The primary critical mechanism driving cell survival is E-cadherin-based cell-cell adhesion and subsequent activation of the PI3K signaling pathway. 195 MATERIALS AND METHODS Cell Culture Human primary prostate epithelial cells (PECs) derived from prostectomy specimens were isolated, cultured, and verified to be free of stromal contamination as described previously (27, 79). Specific patient samples used in this study were again verified to be negative for the stromal cell marker smooth muscle actin by immunostaining. PECs were grown in Keratinocyte—SFM medium (lnvitrogen) supplemented with bovine pituitary extract (BPE) and epidermal growth factor (EGF). Experiments were reproducibly performed in cells derived from two different patients at three different passage numbers (passage 2, 3, and 4). In addition, at least three separate primary cultures from each patient were used. Experiments were verified at least three times for each of the two patients. We were also able to induce differentiation in an immortalized cell line derived from a third patient. The AR-positive prostate cancer cell line LNCaP was purchased from ATCC. LNCaP cells were grown in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum, 2mM glutamine, 50 IU penicillin, 50 uglml streptomycin, 0.225% glucose, 10 mM HEPES, and 1mM sodium pyruvate. Differentiation Assay To induce differentiation, a confluent 10-cm culture dish of PECs was divided equally between three 8-chambered slides (Lab-Tek). Cells were grown in Keratinocyte-SFM supplemented with BPE, EGF, 10 ng/ml keratinocyte growth 196 factor (KGF) (Calbiochem), and 5-10 nM dihydrotestosterone (DHT) (Sigma) for 10-18 days. KGF and DHT were replenished three and five times a week respectively. For larger-scale experiments, three 10-cm plates of confluent PECs were combined onto one 10-cm dish and treated with KGF and DHT for 21-30 days. KGF-blocking Experiments KGF/FGF7 blocking antibody (clone 29522) was purchased from R&D Systems. 2 uglml KGF-blocking antibody or lgG control was added immediately prior to KGF addition. Differentiation of PECs was then assessed by immunofluorescent staining for differentiation markers. Cell Surface lntegn'n and TMPRSSZ Expression Analysis Whole cultures of differentiated PEC cultures were placed in suspension by washing the cells twice with PBS, treating with cell dissociation buffer (Gibco, Invitrogen) for 5 minutes, then adding TrprE Express trypsin (Gibco, Invitrogen). Cells were then washed with wash buffer (1% sodium azide/2°/o FBS-PBS) and incubated with primary antibodies or control lgG molecules for 1 hour at 4°C. Cells were washed twice and incubated with fluorescently-labeled secondary antibodies for 1 hour at 4°C in the dark. Cells were washed twice more, and fluorescence was detected by a Becton-Dickinson FACSCaIibur 4-color flow cytometer with CellQUEST Pro® Software v5.2.1 (Becton-Dickinson). 197 Isolation of Differentiated Cells Differentiated PEC cultures were washed with 1mM EDTA in PBS without calcium or magnesium, and then incubated for 5 minutes with 1mM EDTA/PBS. Cells were then incubated with cell dissociation buffer (Gibco, Invitrogen) for 6-8 minutes. The top layer of cells could then be removed by pipetting the cell dissociation buffer over the cells; the bottom confluent cell layer remains attached to the culture vessel. The isolated cells were used directly or undifferentiated q6-integrin-expressing cells were separated from the differentiated cells using (:6 integrin antibodies and FACS as described above using fluorescently—conjugated integrin (16 antibody (BD Phanningen). Cells were sorted on a Becton-Dickinson FACSAria special order system 12-color flow cytometer using FACSDiVa® software v5.2 (Becton-Dickinson). Immunoblotting Total cell lysates were prepared for immunoblotting as previously described (27, 80). Briefly, cells were lysed with Triton-X 100 lysis buffer, and 45-75 pg of total cell lysates in 2X SDS sample buffer were boiled for 10 minutes. Samples were run on SDS polyacrylamide gels following standard SDS-PAGE protocols and transferred to PVDF membrane. Membranes were blocked in 5% BSA in TBST for two hours at room temperature, then were probed with primary antibody overnight at 4°C. Membranes were washed three times, and incubated with horseradish peroxide-conjugated secondary antibodies (Bio-Rad) in 5% BSA in TBST for 1 hour at room temperature. After washing an additional three times, 198 signals were visualized by a chemiluminescence reagent with a CCD camera in a Bio-Rad Chemi-Doc Imaging System using Quantity One software v4.5.2 (Bio- Rad). Immunoblotting Antibodies Antibodies for phospho-specific Akt (S473) or phospho-specific ERK 1/2 (T202/Y204) were purchased from Cell Signaling. Antibodies for total ERK were from Becton-Dickinson Transduction Labs and total Akt antibodies have been described previously (81). Integrin 06 and TMPRSS2 antibody were gifts from Dr. Anne Cress (University of Arizona, Phoenix, AZ) and Dr. Peter Nelson (Fred Hutchinson Cancer Research Institute, Seattle, WA) (82) respectively. Androgen receptor antibody (441) was purchased from Santa Cruz. E-cadherin antibody (clone HECD1) was purchased from Zymed. Tubulin antibody (clone DM1A) was purchased from Sigma. Immunofluorescence Differentiated PEC cultures were fixed with 4% paraformaldehyde (Mallinckrodt Chemicals) for ten minutes and permeabilized four minutes with 0.2% Triton-X 100 (EMD) at room temperature. Cells were then blocked with 10% normal goat serum (Pierce) for 2 hours at room temperature before incubation with primary antibodies overnight at 4°C. Cells were incubated with appropriate secondary antibodies for one hour at room temperature. DNA was visualized by staining with Hoechst 33258 (Sigma) for 10 minutes at room temperature. Cells were 199 washed three times with PBS between all steps. Coverslips were mounted on the slides using Gel-Mount (Biomeda). Specific antibodies against proteins of interest were obtained as indicated in Table 5 and used for immunofluorescent (IF) staining at the stated dilutions. Whole lgG antibodies for controls were purchased from Pierce. Species appropriate Alexa Fluor 488 or 546 antibodies (Molecular Probes, Invitrogen) were used as secondary antibodies for indirect fluorescence. Table 5. lmmunofluorescence Antibodies IF Protein Clone Dilution Company _goatAb PSA C-19 1:100 Santa Cruz mAb AR 411 1:500 Santa Cruz mAb Bcl-2 100 1:50 Santa Cruz mAb E-cadherin HECD-1 1:100 Zymed/lnvitrogen mAb EGFR Ab12 1:200 Neomarkers mAb ITGA2 P1 H5-E9 1:10 Gift from W.G. Carter mAb ITGA2 PlE6-1-1 1:10 Gift from WC. Carter mAb ITGA3 P1 F2-1-1 1:10 Gift from W.G. Carter mAblTGAV 272-17E6 1 :250 AbCam mAb K18 CY-90 1:100 Sigma mAb K19 A53-BIA2 1:50 Sigma mAb LM5 (72 chain) D4B5 1:100 Chemicon mAb p63 4A4 1:100 Santa Cruz mAb PSA 18127 1:100 R&D Systems mAb PSMA YPSMA-1 1:250 AbCam mAb SMA 1A4 1:100 Zymed/Invitrogen mAbTMPRSSZ P5H9-A3 1:250 Gift of PS. Nelson rAb K5 AF138 1:500 Convance rAb Ki67 1:200 Zymed/lnvitrogen rAb Kip1/p27 G173-324 1:100 Pharmigen rAb ka3.1 H-50 1:500 Santa Cruz ratAb ITGA6 GoH3 1:100 BD Pharrningen Iowa State Univ. Hybridoma ratAb ITGB1 A||B2 1:100 Bank ratAb ITGB4 P4GH-1 1 :10 Gift from WC. Carter 200 Microscopy Epifluorescence images were acquired using a Nikon Eclipse TE300 fluorescence microscope using OpenLab v5.5.0 image analysis software (lmprovision). Confocal images were acquired by sequential detection using a Zeiss 510 Meta NLO v4.2, or Olympus Flroiew 1000 LSM using Flroiew software v5.0. PSA Quantification Differentiated PEC cultures in 8-chambered slides were grown in the presence or absence of DHT for 72 hours in 200 uL per well of growth medium. To quantify PSA concentrations in conditioned medium, a human PSA ELISA kit (Abzyme) was used according to manufacturer’s directions with the following modifications: The entire 200 pl samples were incubated 50 ul at a time per well for 1 hour each. PSA standards were added to coated wells during the final 50 pl of sample incubafion. Reverse Transcription PCR (R T—PCR) for Differentiation Markers Human KLK2, human KLK3 (PSA), FGFR2Illb, and GAPDH mRNA levels were quantified in differentiated cells by RT-PCR. Total RNA was isolated from upper and lower cell populations of dissociated cells from differentiated cultures or from LNCaP cells using TRIzol (Gibco) and chloroform (Sigma-Aldrich). Contaminating DNA was then removed using RNAse-free DNAse kit (Qiagen) following manufacturer’s directions. RT-PCR was performed on 1-2 pg RNA with 201 the primers listed in Table 6 using the One-Step RT-PCR kit (Qiagen) following manufacturer’s directions. RT-PCR products were analyzed on a 2% agarose/T BE gel and DNA was visualized with ethidium bromide and a CCD camera in a Bio-Rad Chemi-Doc Imaging System using Quantity One software v4.5.2 (Bio-Rad). TABLE 6: RT-PCR Primers Target Fwd Primer (5’—>3’) Rev Primer (5’—>3’) Ref ITGA6 GCTGGTTATAATCC'I'I'CAATA TTGGGCTCAGAACCTT (83) TCAA'I‘I’GT GGTTT ITGB1 GTGGTTGCTGGAA‘I'I’GTTCT | l l ICCCTCATAC'I'I'C (83) TATT GGATTGAC AR | | l ICAATGAGTACCGCATG TCTCGCAATAGGCTG (84) C CACG FGFR2III ATTG'I'I'CTCCTGTGTCTG Cl l I ICAGC'ITCTATA (85) b TCC GAPDH ACCACAGTCCATGCCATCAC TCCACCACCCTGTI'G (86) CTGTA KLK2 GGCAGGTGGCTGTGTACAGT CAACATGAACTCTGTC (87) C ACC'I‘I'CTC KLK3 CCCACTGCATCAGGAACAAA GGTGCTCAGGGG'I‘I’G (87) (PSA) AGCG GCCAC Small Interfering RNA Transfections A pool of four small interfering RNAs (siRNA) against androgen receptor (siGENOME SMARTpool) or a non-targeting sequence were purchased from Dharmacon. Differentiated cultures were transfected with 20 nM siRNA in keratinocyte-SFM medium using siLentFect lipid reagent (Bio-Rad) and Opti- MEM (lnvitrogen) medium following manufacturer’s directions. The medium was changed 16 hours after transfection. 202 Cell Survival Assays Differentiated PECs were growth factor-starved in keratinocyte-SFM medium containing no supplements, KGF, or DHT for 72 hours. Then DMSO (control; Sigma), pharmacological inhibitors 0.5 uM PD168393, 2 uM LY294002, 20 uM P090859, 10 uM SB209102, 10 uM 420119 (all purchased from Calbiochem), 1 uM staurosporine (Promega), or 1 ug/ml E-cadherin blocking antibody (SHE78-7, Calbiochem) or non-specific mouse lgG (Sigma) was added; in some experiments, siRNAs were used to knock down AR expression (Dham'racon). Cells were incubated for 24, 48, 66, or 72 hours after drug, antibody, or siRNA addition. LY294002 was replenished 48 hours after its initial addition. To assess cell viability, cells were fixed and DNA fragmentation was monitored using Terminal Deoxynucleotide Transferase dUTP Nick End Labeling (T UNEL) following the protocol of the APO-BrdU TUNEL Assay Kit (BD Phanningen). On several occasions, cleaved caspase 3 (Asp175) staining with antibody clone 5A1 from Cell Signaling was also used to measure cell viability of fixed cells. TUNEL and caspase activity were quantified using Imagine software (88).. Total TUNEL- or caspase-positive pixels were normalized to total propidium iodide-stained DNA pixels in fixed cells and expressed as relative intensity of TUNEL staining. This quantification is based on pixel counts and does not necessarily reflect the percentage of positive cells, but rather the relative intensity of TUNEL or caspase 3 staining between treated and untreated cultures. As an alternative method for measuring cell viability, unfixed cells were treated with propidium iodide (Pl). 203 High intensity PI staining of dead, i.e. permeabilized cells, was quantified on a per cell basis and expressed as % Pl positive cells. ACKNOWLEDGEMENTS We thank Veronique Schulz, Rich West, and Dr. Jim Resau at the Van Andel Research Institute (VARI) and Dr. Melinda Frame at Michigan State University for technical assistance, and we acknowledge members of the laboratories of Developmental Cell Biology, Systems Biology, and Cell Structure and Signal Integration at VARI for their constructive suggestions. We also thank Dr. Doug Green (St. Jude Children’s Research Hospital, Memphis, TN) for his constructive suggestions on the cell survival analyses. Integrin 02, a3 and 84 antibodies where a kind gift of Dr. William Carter (Fred Hutchinson Cancer Research Institute, Seattle, WA); the TMPRSSZ antibody was a generous gift of Dr. Peter Nelson (Fred Hutchinson Cancer Research Institute, Seattle, WA) and the (:6 integrin blotting antibody was a gift from Dr. Anne Cress (University of Arizona, Phoenix, AZ). 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Similar to primary prostate cancer, there was an increase in integrin (1681 with a simultaneous decrease in the other integrins. Like metastatic castrate-resistant prostate cancer, these cells were not dependent on androgen for AR-mediated phenotypes. While this model has several limitations inherent from its parental line and the caveat that AR is being re-expressed in these cells, it is currently one of the better models to use in the context of understanding the relationship between AR and integrin (:6 in CRPC. AR-dependent phenotypes can be elucidated without the complication of endogenous AR, siRNA against AR can be used to validate and directly test AR- dependent effects, AR mutants can be expressed to refine mechanisms, and the cells express the Iaminin integrin 0681, which is the predominant integrin in primary prostate cancer. We also developed a novel in vitro differentiation model in which normal human primary prostate basal epithelial cells isolated from patients can be induced to differentiate into AR-expressing and androgen responsive cells that recapitulate many of the in vivo characteristics of secretory cells (1). To our knowledge, this is the first in vitro differentiation model in which sufficient numbers of AR-expressing prostate cells can be generated for experimental investigation. These cells can be treated with pharmacological 215 inhibitors and siRNA to study bioChemical and genetic effects. Furthermore, the differentiated AR-expressing cells can be isolated for further analysis. Using these and other models, we have identified some of the pathways required for survival of prostate and prostate cancer cells (summarized in Table 7). Previous studies from our lab have demonstrated that in normal prostate basal epithelial cells, adhesion to Iaminin 5 via 0381 to ERK through EGFR or through Src is required for cell survival (2). The PI3K/Akt pathway is not activated on LM5 and not required for survival. Disruption of either the EGFR/ERK or Src pathway leads to caspase-independent cell death, due to the generation of reactive oxygen species (2). We have demonstrated that in differentiated, AR-expressing prostate epithelial cells, while KGF, AR, and androgen are important for initiating the differentiation process and AR is important to maintain the androgen-dependent phenotype of secretory-like cells, these factors are not required for survival of the committed differentiated cells (1). The primary critical mechanism driving differentiated cell survival is E-cadherin- based cell-cell adhesion and subsequent activation of the Pl3K signaling pathway (1). We have further demonstrated that in two prostate tumor cell lines, PC3 and DU145, PI3K signaling is required for cell survival. We and others have also shown PI3K signaling to be required for survival in LNCaP cells. In addition, adhesion of P03 cells to Iaminin 1 promotes survival via integrin-mediated activation of Src leading to increased expression of the pro-survival protein Bcl- xL (2). Inhibition of PI3K or Src results in caspase-dependent cell death (2). In 216 DU145 cells, adhesion to collagen 1 drives survival through activation of EGFR and ERK. Re-expression of AR in PC3 cells can rescue cells from death induced by inhibition of PI3K when adherent to Iaminin 1. Expression of AR in PC3 cells leads to increased expression of integrin 0681 and Bcl-xL along with increased activation of NF-xB. Blocking each of these components individually concurrent with inhibition of PI3K led to death of the AR-expressing cells, suggesting that AR regulates cell survival through enhancement of 0681/NF- KB/Bcl-xL signaling. Keeping in mind the caveat that different cell lines were used and that the survival pathways of these cells were studied on different matrices, several broad conclusions can be drawn from this data. First, all of the prostate cancer cell TABLE 7. Summary of AR, PTEN, and Integrin «6 Expression and Survival Pathways in Prostate and Prostate Cancer Cell Lines In Vitro Normal Tumor Basal (PEC) Differentiated PC3 DU145 LNCaP PC3-AR § AR No Yes No No Yes Yes g PTEN Yes n.d. No Yes No No é ITGA6 Moderate No High Moderate Low Highest ECM-Cell Adhesion (Integrin) Yes No Yes Yes Yes Yes Cell-Cell _ Adhesion 3 (E-cadherin) n.d. Yes No n.d. n.d. No 0;) EGFR Yes No No Yes n.d. n.d. ERK Yes No No Yes n.d. n.d. PI3K No Yes Yes Yes Yes Yes* Src Yes n.d. Yes n.d. n.d. No AR No No No No Yes Yes* Chapter (Reference) Ch. 1 (2) Ch. 4 (1) Ch. 2 (2) Ch. 2 Ch. 2 (3) Ch. 3 * Inhibition of both pathways required to induce cell death 217 lines tested were dependent on PI3K signaling. While the PC3 and LNCaP cell lines do not express Pten (a negative regulator of PI3K signaling) and therefore their dependence on PI3K for survival might be expected; the DU145 cells do express Pten, but still depend on Pl3K for survival. Integrin-mediated activation of PI3K/Akt suggests that Akt activity can still be elevated in prostate cancer in the absence of Pten loss. Furthermore, targeting Pl3K may still be an effective therapy in tumors that retain Pten expression. Second, PEC cells are not dependent on PI3K signaling, but differentiated AR-expressing cells are. This suggests that prostate cancer may arise from a more differentiated cell that has acquired dependence on PI3K for its survival and expresses AR. Third, while PEC cells are dependent on integrin-mediated adhesion to matrix and subsequent activation of EGFR for survival, differentiated AR-expressing cells do not express integrins, are not adherent to LM or CL matrix, and have reduced levels of EGFR. Subsequently, the cells are not dependent on EGFR or matrix adhesion for survival and instead are dependent on cell-cell adhesion and PI3K to promote cell survival. Fourth, PC3 cells adherent to collagen rather than Iaminin were not dependent on PI3K signaling for survival. Since the primary metastatic site for prostate cancer in humans is to bone which consists mostly of collagen, this has interesting implications for designing therapies for advanced disease (i.e. targeting Pl3K signaling may not be effective in bone metastasis). DU145$ are not as invasive and metastatic as PC3 cells, perhaps representing an intermediate phenotype between PEC and PCB cells. In support of this, DU145 cells, like PEC cells, were dependent on EGFR for survival. PC3 218 cells were not dependent on this pathway for cell survival. Interestingly, both PEC and PC3 cells were dependent on Src for survival, suggesting that maintenance of this pathway is critical during disease progression. However, expression of AR in the PC3 cells significantly altered the survival pathways in PC3 cells. Loss of Src or PI3K signaling alone was no longer sufficient to induce cell death. Rather, loss of AR expression or one of its downstream pro-survival targets (i.e. integrin 06, NF-ch, or BcI-xL) was also required to restore dependence on P|3K signaling for survival. Thus, AR-expressing cells have a distinct survival advantage over AR-negative cells. Furthermore, this supports the need for combinational therapies in order to target and kill prostate cancer cells. Validation of AR Effects in Additional Models Other Cell Lines Although we have established a relationship between AR, integrin 06, NF- KB, and Bcl-xL in PC3-AR cells, the PC3-AR model is not perfect, in that it did not originally express AR, has lost Pten expression, and only represents one oncogenic profile that could be important in this disease. Therefore, it is necessary to extend these studies to additional cells lines in order to generalize whether or not these relationships exist in other cell lines and determine, if oncogenic status (i.e. expression of Pten) influences this relationship, if endogenous AR has the same effects as exogenously expressed AR, and if there is an effect due to androgen sensitivity. We have already demonstrated 219 some of these relationships in LNCaP cells, which express endogenous but a more promiscuous and mutated AR. We have recently acquired C4-2B cells, a derivative of LNCaP cells that have lost their androgen sensitivity but still retain AR expression. Furthermore, we have re-expressed AR in DU145. Fortunately, some additional new cell lines that express endogenous AR and have different oncogenic backgrounds have also become available, including VCaP, LAPC4, MDA PCa 2b, and 22Rv1 cells. We are currently characterizing relative AR, integrin 06, and Bcl-xL expression levels as well as NF-xB activity in these cells. Lastly, the ability to differentiate primary human prostate epithelial cells needs to be tested in cells derived from other patients as well as from other sources. In Vivo Studies In addition to investigating AR and integrin survival pathways in other cell lines, we also need to establish whether they are important in vivo. We are currently investigating existing clinical microarray data sets to determine if a correlation exists between integrin 06 and prostate cancer progression to CRPC. Furthermore Jelani Zarif, a fellow graduate student in the lab, has developed lentiviral constructs that express tetracycline-inducible shRNAs against AR and has demonstrated their effectiveness in vitro. These will be used to inhibit components of the AR survival pathway in PC3 cells and their ability to form tumors when injected orthotopically into nude mice. Castration and PI3K inhibitor studies individually or in combination will be used to test the importance of the pathways in tumor survival. The tumors will also be analyzed for expression of 220 AR, integrin 06, and Bcl-xL as well as NF-xB or caspase activity by either immunohistochemistry or immunoblotting to validate activation of the pathways in vivo. Understanding the Process of Differentiation in Prostate Epithelial Cells Using the in vitro differentiation model we have generated, we can now begin to interrogate the processes that are required and sufficient to drive differentiation. Preliminary data from our lab suggests that inhibition of p38 signaling, but not EGFR or ERK, inhibits the bilayer formation, suggesting that p38 plays an important role in early differentiation. One of the most dramatic observations was loss of the 72 chain of LM5. This appeared to occur prior to integrin [31 loss. We still need to determine if this is observed for other LM chains and other matrices as well. Is loss of LM5 driving loss of integrin expression, or is integrin lost through another mechanism? Furthermore, what is the mechanism driving LM5 loss? Is it by regulation of the LM5 chains directly, or indirectly through secretion of matrix metalloproteinases (MMPs)? Is loss of LM5 sufficient to drive early stages of differentiation? These questions can be tested in part by using LM5 specific siRNA, qRT-PCR, and zymography to test for the presence of MMPs. Next Generation of AR-expressing Models The differentiation model we have described can now be further optimized for generating AR-expressing cells. It may be that pre-selecting cells with “stem- 221 cell” characteristics before treating with KGF and DHT may enhance this process (4-6). Since KGF is a stromal-derived growth factor, it is exciting to postulate that culturing with additional stromal factors or by performing co-cultures with stromal cells may help further differentiate these cells to appear more columnar in appearance and have increased nuclear AR localization and function. Also, using the culture conditions we have described, it would be interesting to see if this would allow for culturing of human AR-expressing primary prostate cells, since an in vitro model for these cells do not exist. Another approach to generate a model of primary prostate cancer may be to genetically alter the differentiating cells to become tumorigeneic. In the lab, we have already generated immortalized PEC cells from two patients by expressing the viral oncogenes E6/E 7 and hTERT. Using the method described, we can induce differentiation in these cells as measured by AR, PSA, and K18/19 positivity. Interestingly, subsets of these differentiated cells retain integrin B1 expression. The co-expression of AR and integrins in the same cells suggests that genetic manipulation of this model is possible. Since patients do not develop prostate cancer from E6/E7 and hTERT overexpression, we would also want to immortalize or transform PEC cells using oncogenic mutations associated with prostate cancer progression. This includes c-Myc and TMPRSSZ/Erg overexpression, or loss of the tumor suppressor proteins Pten and p27/Kip1. Development of this differentiation model may allow us to test potential prostate cancer therapeutics to see if they will be toxic in normal prostate 222 epithelial cells, develop a model for early prostate cancer tumorigenesis, and once a number of different patient samples are tested and characterized, may be able to develop a model for slow growing, benign cancer versus faster growing aggressive cancer. Therapeutic Implications This data supports the concept that more than one pathway may need to be targeted in cancer in order to effectively target and kill the cells. This has been effective in the treatment of other diseases, such as HIV where a combination of three or four anti-retroviral drugs can provide years of extended life. This has been demonstrated in mouse models of prostate cancer where targeting AR and mTOR, a downstream target of PI3K/Akt signaling, has additive effects in reducing tumor size (7). Based on our studies, we propose that targeting AR, integrin o6, NF-ch, and/or PI3K/Akt may be an effective combination for advance metastatic prostate cancer. Drugs targeting NF-KB and PI3K/Akt are currently available or are in phase II" clinical trials (reviewed in 8, 9). Integrin qu3 blocking antibody has been tested in clinical trials (10-12) and intratumoral administration of Iiposome-encapsulated integrin ov-siRNA has been shown to inhibit prostate tumor growth in bone xenografts (13). Systemic administration of integrin av has had some side effects, perhaps due to its role in angiogensis (10-12, 14). However, we would predict that blocking integrin 06 would not have toxic side effects, at least in the prostate and in skin since both these cell types do not depend on integrin (16 for survival and adhesion to Iaminin 223 can also be mediated by integrin 0381 (2, 15). In addition to ADT drugs, AR may be targeted directly. Recently, pre-clinical studies have demonstrated that adeno-associated virus (AAV)-delivery of shRNA specific for AR can reduce tumor burden of human prostate cancer xenografts in nude mice (16). Although AAV has been successfully utilized for gene therapy in some phase II” trials, there are some concerns that this can lead to insertional mutagenesis and immunogenesis (17-19). This makes nanoparticles an attractive alternative, where multiple drugs or RNAi’s targeting AR, integrin d6, NF-KB, and/or PI3K/Akt could be attached (20). Nanoparticles have already shown to be well tolerated in animal models and effective at targeting prostate cancer cells in xenograft models (21). It may be that by targeting several pathways, lower levels of drugs could be used. Using an integrin o6 recognizing antibody would have the additional advantage of targeting the nanoparticles to prostate cancer cells. 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