Function of Androgen Receptor in Prostate Cancer Epithelial Mesenchymal Transition and Microtubule Targeting

University of Kentucky UKnowledge

University of Kentucky Doctoral Dissertations Graduate School

2010

FUNCTION OF ANDROGEN RECEPTOR IN PROSTATE CANCER EPITHELIAL MESENCHYMAL TRANSITION AND MICROTUBULE TARGETING

Menglei Zhu University of Kentucky, [email protected]

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Recommended Citation Zhu, Menglei, "FUNCTION OF ANDROGEN RECEPTOR IN PROSTATE CANCER EPITHELIAL MESENCHYMAL TRANSITION AND MICROTUBULE TARGETING" (2010). University of Kentucky Doctoral Dissertations. 109. https://uknowledge.uky.edu/gradschool_diss/109

This Dissertation is brought to you for free and open access by the Graduate School at UKnowledge. It has been accepted for inclusion in University of Kentucky Doctoral Dissertations by an authorized administrator of UKnowledge. For more information, please contact [email protected]. ABSTRACT OF DISSERTATION

Menglei Zhu

The Graduate School University of Kentucky 2010 FUNCTION OF ANDROGEN RECEPTOR IN PROSTATE CANCER EPITHELIAL MESENCHYMAL TRANSITION AND MICROTUBULE TARGETING

______

ABSTRACT OF DISSERTATION ______

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Medicine at the University of Kentucky

Menglei Zhu Lexington, Kentucky

FUNCTION OF ANDROGEN RECEPTOR IN PROSTATE CANCER EPITHELIAL MESENCHYMAL TRANSITION AND MICROTUBULE TARGETING

Prostate cancer is the most frequently diagnosed non-skin cancer and the third leading cause of cancer mortality among men in the US. Androgens are functionally required for the normal growth of the prostate gland and play a critical role in prostate tumor development and progression. Epithelial-mesenchymal-transition (EMT) is an important process during normal development, and cancer cell metastasis. This study examined the ability of androgens to influence EMT of prostate cancer epithelial cells and evaluate the effect of taxol chemotherapy on androgen signaling in prostate cancer cells in prostate cancer. The EMT pattern was evaluated on the basis of expression of the epithelial markers as well as cytoskeleton reorganization in respond to DHT (1nM) and/or TGFβ (5ng/ml). Overexpressing and silencing approaches to regulate androgen receptor (AR) expression were conducted to determine the involvement of AR in EMT in the presence or absence of an AR antagonist. The AR transcriptional activity was determined on the basis of prostate specific antigen (PSA) mRNA expression and the androgen-response element (ARE) luciferase reporter assay. The interaction of AR and tubulin was investigated using immunoprecipitation, immunofluorescence as well as introduction of a truncated AR in human prostate cancer cells. Our results demonstrate that androgens induce the EMT pattern in prostate tumor epithelial cell with Snail activation and led to significant changes in prostate cancer cell migration and invasion potential. Expression levels of AR inversely correlated with androgen-mediated EMT in prostate tumor epithelial cells, pointing to a low AR content required for the EMT phenotype. Our study also reveals that treatment of prostate cancer cells with Paclitaxel or Nocodaxol inhibits androgen-dependent, as well as androgen- independent AR nuclear translocation and activation potentially via targeting the interaction of AR and microtubule cytoskeletal structures. Our findings on multiple aspects of AR function in prostate cancer development and progression may enhance the understanding of AR targeting therapy being a double-sided sword in the context of tumor microenvironment. These studies provide new insights into the mechanism of action of chemotherapy agents and the development of therapeutic resistance within tubulin/microtubule repertoire in prostate cancer cells. KEYWORDS: prostate cancer, androgen receptor, EMT, taxol, chemotherapy

Menglei Zhu Student’s Signature

May 6, 2010 Date FUNCTION OF ANDROGEN RECEPTOR IN PROSTATE CANCER EPITHELIAL MESENCHYMAL TRANSITION AND MICROTUBULE TARGETING

By Menglei Zhu

Natasha Kyprianou, Ph.D. Director of Dissertation

David Orren, Ph.D. Director of Graduate Studies

May 6, 2010 Date

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Name Date

DISSERTATION

Menglei Zhu

The Graduate School University of Kentucky 2010 FUNCTION OF ANDROGEN RECEPTOR IN PROSTATE CANCER EPITHELIAL MESENCHYMAL TRANSITION AND MICROTUBULE TARGETING

______

ABSTRACT OF DISSERTATION ______

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Medicine at the University of Kentucky

Menglei Zhu Lexington, Kentucky

Director: Dr. Natasha Kyprianou, Professor of Surgery/Urology, Molecular and Cellular Biochemistry, Toxicology Lexington, Kentucky 2010 Copyright © Menglei Zhu 2010 This dissertation is dedicated to my mother, Ms. Yiqun Liu.

ACKNOWLEDGEMENTS

I would like to begin by thanking to my mentor, Dr. Natasha Kyprianou, whose encouragement, supervision and support from the preliminary to the concluding level enabled me to develop an understanding of the subject. I would not have finished this thesis without the personal and professional support of her. She is a warm and caring person with such an intriguing scientific mind. What I learned from her is not only the science itself, but more importantly, the passion for science. I would also like to thank all the members of my committee, Dr. Daret St. Clair, Dr. Steven Schwarze and Dr. Haining

Zhu. I also want to thank Dr. Craig Horbinski for his professional assistance for the pathological evaluation of patient tissue sample and valuable suggestions for my paper. I am grateful for their critical feedback over the years. Their contributions have made me a better scientist. I also want to thank Dr. Mary Vore, director of Toxicology and Dr.

David Orren, our DGS for the support during my whole graduate study especially my hard time.

While being a member of the Kyprianou lab, I have been fortunate to work with a number of quality scientists who have helped me greatly along the way. I thank Dr. Brain

Zhu for numerous valuable discussions and walking me through countless valuable scientific techniques, Dr. Hong Pu for assistance of immunochemistry staining and image evaluation, Dr. Shinichi Sakamoto for his work with the cytoskeleton and integrin and

Joanne Collazo for her help on the daily work. But most importantly, I would like to thank them all for being my friends and putting up with me all these years.

I'd like to thank our collaborators who provided me the important materials for

my research: Dr. Tom Beer, Dr. Mark Garzotto and Dr. David Qian from Oregon Health

iii

and Science University; Dr. Don Tindall from Mayo Clinic; Dr. Kerry Burnstein from

University of Miami; and Dr. Zoran Culig from University of Innsbruck.

I've been fortunate to have the emotional support of my family and friends that I

carried with me every day. I am grateful for my fiancé, Anyi, who has never been angry

with me for not being around, tolerant of my bad temper when my project did progress

well, and makes sure I alleviate my stress often. Finally, I am thankful for my idol, the

strongest woman I have ever known, my mother. She has always believed in me,

supported me, and given me everything I could ever want or need. Lastly, I offer my regards and blessings to all of those who supported me in any respect during the completion of the project.

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ...... iii TABLE OF CONTENTS ...... v LIST OF TABLES ...... vii LIST OF FIGURES ...... viii CHAPTER ONE ...... 1 INTRODUCTION ...... 1 The Prostate Gland ...... 1 Epidemiology of Prostate Cancer ...... 3 The Androgen Receptor and its Signaling Partners ...... 3 AR Interaction with Key Growth Factor Signaling Pathway ...... 4 AR and TGFβ: Death Links Life Partners ...... 10 AR Plays in the Stroma: Functional Promiscuity ...... 11 Summary ...... 13 CHAPTER TWO ...... 21 EXPERIMENTAL DESIGN AND METHODS ...... 21 Cell Lines ...... 21 Antibodies and Chemicals ...... 21 Transfections ...... 22 Wounding Assay ...... 22 Flow Cytometry Analysis ...... 22 Western Blot Analyses ...... 23 Immunofluorescence Staining ...... 23 RNA Extraction and Realtime RT-PCR ...... 24 Patient Population ...... 24 Specimen Collection and Specimen Processing ...... 24 Tissue Microarray Construction ...... 25 Immunocytochemical Staining ...... 26 Immunoprecipitation Analysis ...... 26 Luciferase Activity Assays ...... 27 Statistical Analysis ...... 27 CHAPTER THREE ...... 28 ANDROGREN RECEPTOR CROSSTALK WITH TGFβ SIGNALING PATHWAY .. 28 Introduction ...... 28 TGFβ Regulates AR-dependent Gene Expression in Prostate Cancer Cells ...... 29 Impact of TGFβ and DHT Interaction on TGFβ mediated Transcription ...... 30 TGFβ Increases AR Nuclear Translocation Activity ...... 30 DHT and TGFβ Promote AR-Smad4 Association ...... 31

Apoptosis Outcomes in Response to TGFβ and DHT in PC3 Prostate Cancer Cells Expressing Wild-type AR ...... 31 CHAPTER FOUR ...... 43 EFFECT OF ANDROGEN AND ANDROGEN RECEPTOR ON EPITHELIAL- MESENCHYMAL TRANSITION (EMT) IN PROSTATE CANCER CELLS ...... 43 Introduction ...... 43 Results ...... 45 Discussion ...... 49 CHAPTER FIVE ...... 70 CHEMOTHERAPY-BASED MICROTUBULE TARGETING SUPPRESSES ANDROGEN RECEPTOR ACTIVITY IN PROSTATE CANCER CELLS ...... 70 Introduction ...... 70 Results ...... 72 Discussion ...... 75 CHAPTER SIX ...... 99 DISCUSSION ...... 99 Androgen Axis Cross-Talk with TGFβ Signaling in Prostate Tumorigenesis ...... 99 Androgen Receptor Signaling and Tumor Microenvironment ...... 101 Targeting EMT: Potential Clinical Value in Advanced Prostate Cancer ...... 103 Microtubules Facilitate AR Nuclear Translocation and Transcriptional Activity ...... 104 Combination of Taxol Chemotherapy with Androgen Deprivation Therapy ...... 105 Androgens and AR: Key Regulators in Prostate Cancer Progression ...... 106 Future Directions ...... 108 REFERENCES ...... 110 Vita ...... 122

LIST OF TABLES

Table 2. 1 Cell lines ...... 20 Table 4. 1 Molecules Involved in EMT ...... 69

vii

LIST OF FIGURES

Figure 1. 1 The Anatomy of the Prostate Gland ...... 14 Figure 1. 2 AR Signaling Pathway ...... 16 Figure 1. 3 Growth Factors Crosstalk with AR in Prostate Cancer Cells...... 18 Figure 3. 1 Effect of TGFβ and DHT on Probasin Luciferase Activity and PSA Expression in LNCaP TβRII Cells...... 33 Figure 3. 2 Regulation of TGFβ Transcriptional and Apoptotic Activity by Androgens. 35 Figure 3. 3 Effect of TGFβ and DHT on AR Nuclear Translocation in LNCaP-TβRII Cells...... 37 Figure 3. 4 Combination of DHT and TGFβ Results in AR-Smad4 Association in Prostate Cancer Cells...... 39 Figure 3. 5 Effect of TGFβ and DHT in PC3-AR Cells...... 41 Figure 4. 1 Effect of Androgens on EMT of Prostate Cancer Epithelial Cells...... 51 Figure 4. 2 Androgens Regulate Cytoskeleton Reorganization of Prostate Cancer Cells...... 53 Figure 4. 3 Effect of Androgens and TGFβ on Prostate Cancer Cell Invasive Behavior. 55 Figure 4. 4 Detection of AR in PC3 Cells...... 57 Figure 4. 5 AR Status and EMT...... 59 Figure 4. 6 AR Involvement in EMT-related Cytoskeleton Reorganization and Cell Invasion...... 61 Figure 4. 7 Effect of Non Genomic-BSA-conjugated Testosterone on EMT Signaling in Prostate Cancer Cells...... 63 Figure 4. 8 Overexpression of AR inhibits PC3 cell viability...... 65 Figure 4. 9 Androgens Induce EMT in Human Breast Cancer Cells but Not in Renal Carcinoma Cells...... 67 Figure 5. 1 Doclitaxel Suppresses PSA Expression in Human Prostate Tumors ...... 78 Figure 5. 2 Microtubule Targeting Drugs Inhibit Ligand-dependent AR Transcriptional Activity ...... 80 Figure 5. 3 Microtubule Targeting Inhibits Ligand-independent AR Transciptional Activity ...... 82 Figure 5. 4 Doclitaxel Suppresses AR Nuclear Translocation ...... 84 Figure 5. 5 Tubulin Interacts with AR ...... 86 Figure 5. 6 Androgens Inhibit Tubulin Expression in Prostate Cancer Cells ...... 89 Figure 5. 7 Suppression of Tubulin Expression by DHT and/or TGF β Treatment ...... 91 Figure 5. 8 Expression Pattern of EMT Markers ...... 93 Figure 5. 9 Expression Pattern of Cell Cycle Markers ...... 95 Figure 5. 10 Microtubule Facilitate AR Nuclear Translocation in Prostate Cancer Cells97

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ABBREVIATION

AR, androgen receptor ADT, androgen deprivation therapy AF-1, transcription-activation function-1 ARE, androgen response element BPH, benign prostatic hyperplasia CSS, charcoal-stripped-serum DHT, dihydrotestosterone DBD, DNA-binding domain EGF, epidermal growth factor EMT, epithelial-mesenchymal transition; FGF, fibroblast growth factor EGFR epidermal growth factor receptor HRP, horse-radish peroxidase IGF-1, insulin-like growth factor-1 LBD, ligand-binding domain LUTS, lower urinary tract symptoms Lgl, Lethal giant larvae LNCaP-TβRII, LNCaP TGFβ Receptor II mt AR, mutant androgen receptor MAPK, Mitogen-activated protein kinase MEKK1, MAPK/extracellular signaling-regulated kinase kinase-1 PARP, Poly (ADP-ribose) polymerase PATJ, Pals1-associated tight junction proteins PSA, prostate-specific antigen PCRP, castration resistant prostate cancer PI3K, phosphatidylinositol 3-OH kinase Pca, Prostate cancer PIN, prostatic intraepithelial neoplasia PSMC, prostate smooth muscle cell PS-1, prostate stromal cell SMA, smooth muscle actin SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis TβRII, TGFβ receptor II TNF, Tumor necrosis factor TGF-β, transforming growth factor- β; TRAIL, TNF related apoptosis-inducing ligand VEGF, vascular endothelial growth factor wtAR, wild-type androgen receptor Zeb1, Zinc finger E-box-binding homeobox 1

CHAPTER ONE INTRODUCTION

The Prostate Gland The prostate gland is located at the base or outlet of the urinary bladder and surrounds the first part of the urethra. (Fig 1.1) The primary function is to secrete most of the fluid in the semen, including a proteinaceous substance, minerals and sugar during ejaculation. Another function of the prostate gland is to help control urination by pressing directly against the part of the urethra that it surrounds. The normal prostate gland of a young man is the size of a walnut; as people get older, the gland usually grows larger. The prostate is composed of tubuloalveolar glands arranged in lobules surrounded by a stoma. The prostate gland is rich in nerves, smooth muscle, collagen and lymphatics. Four regions made up this walnut shaped gland: the transition zone, central zone, peripheral zone and anterior fibro-muscular zone. The transition zone surrounds the prostate urethra and comprises 5% of the glandular tissue. BPH and 20% prostate cancer arise here. The central zone surrounds the ejaculatory ducts as they course form the base of the prostate to the verumontanum. It comprises 20 to 25% of the gland and gives rise to 5-10% of prostatic cancers. The peripheral zone lies posteriorly and laterally in the prostate, comprises 70 to 75% of the gland and surrounds the central zone; 70% adenocarcinomas arise from this area. The peripheral zone is the palpable portion of the prostate on digital rectal examination. Periurethral glands lie adjacent to the urethra and are surrounded by the proximal sphincter. Carcinoma does not arise from these glands. The fibromuscular stroma occupies the anterior surface of the prostate and is principally comprised of smooth muscle (NAZ 1997) . The mesoderm-derived epithelial cells of the sex cords in developing testes become the Sertoli cells which will function to support sperm cell formation. Leydig cells, a minor population of non-epithelial cells appears between the tubules by week eight of human fetal development. Soon after they differentiate, Leydig cells begin to produce androgens. The growth-promoting effects of androgens are mediated mostly through the androgen receptor (AR). Androgen action functions through an axis involving the

testicular synthesis of testosterone, its transport to target tissues, and the conversion by 5 -reductase to the more active metabolite 5 -dihydrotestosterone (DHT). Testosterone and DHT exert their biological effects through binding to AR and inducing AR transcriptional activity. The 5 -reductase enzyme is present in the urogenital sinus before and during prostate development (Siiteri and Wilson 1974; Heinlein and Chang 2002). In individuals lacking a functional 5 -reductase gene, the prostate is small or undetectable. Inhibition of 5 -reductase during fetal development results partial prostate development in rats (Imperato-McGinley, Binienda et al. 1985). After the development of the prostate gland, androgens continue to promote survival of the secretory epithelial cells, the primary cell type involved in the malignant transformation to prostate adenocarcinoma (De Marzo, Nelson et al. 1998). There are two common diseases associated with prostate gland: lower urinary tract symptoms (LUTS) which make up a lot of prostate problems, and cancer. Benign Prostate hypertrophy (BPH) is the most common cause of LUTS. BPH is a benign prostate condition that is not associated with prostate cancer. The prostate cancer can also cause similar problem in older men by blocking the outlet of the bladder or the urethra and leads to difficulty with urination. The symptoms commonly include slowing of the urinary stream and urinating more frequently, particularly at night. Prostate cancer is a malignant tumor that consists of glandular epithelial cells from the prostate gland. The tumor usually grows slowly and remains confined to the gland for many years. During this time, the tumor produces little or no symptoms or outward signs. As the cancer advances, however, it can spread beyond the prostate into the surrounding tissues and also can metastasize throughout other areas of the body, such as the bones which is the most favorite metastasis site of prostate cancer. In the initial stages, prostate cancer development and growth is dependent on androgens and can be suppressed by androgen ablation monotherapy (Wang, Yin et al. 2007); due to the emergence of androgen-independent prostate cancer cells, prostate tumors recur as hormone-refractory and highly metastatic for which no treatment is currently available.

Epidemiology of Prostate Cancer Prostate cancer is the most frequently diagnosed non-skin cancer and the third leading cause of cancer mortality in men. It is estimated 186,320 new cases and 28,660 deaths from prostate cancer in the United States in 2008. It is estimated that, in Western countries, about 30% of all men will develop microscopic prostate cancer during their lifetime. However, as most prostate cancers tend to grow slowly, the risk of developing overt clinical disease is 8% (lifetime risk), and the risk of actually dying from prostate cancer is only 3%, whereas the autopsy based prevalence is 80% by the age of 80 years. Therefore, most men die with prostate cancer, rather than from it. The risk factors of prostate cancer include age, ethnicity, family history as well as nutrition. Age is the main risk factor for prostate cancer. It is rare in men younger than 45. Most men with prostate cancer are older than 65. Prostate cancer is more common in African American men than in white men, including Hispanic white men and when diagnosed with prostate cancer present with a higher stage. Overall African-Americans have a poorer survival tate compared with European-Americans. (Wideroff, Schottenfeld et al. 1996) It is less common in Asian and American Indian men, whereas member of Scandinavian countries have a higher incidence. Several studies have demonstrated an increased risk with familial aggregation of prostate cancer. The epidemiology and features of family and hereditary prostate cancer have been described: The familial type was characterized by increased risk of prostate cancer, increased number of affected relatives and earlier age of disease onset. The hereditary type, a subset of the familial group, was characterized by autosomal dominant inheritance form either parent, predisposing men to early development of prostate cancer. They postulated that hereditary type causes up to 9% of prostate cancer(Carter, Bova et al. 1993).

The Androgen Receptor and its Signaling Partners Androgen functions through an axis involving testicular synthesis of testosterone, conversion by 5 -reductase to the active metabolite 5 -dihydrotestosterone (DHT) and its binding to Androgen receptor (AR) to induce transcriptional activation of target genes. Thus 5 -reductase drives the development of prostate gland (Siiteri and Wilson 1974;

Imperato-McGinley, Binienda et al. 1985; Heinlein and Chang 2002). AR is an 919 amino acid protein which bind with HSP90 in cytosol. The binding of ligand (androgen) allows AR to be released from HSP90 and translocated to nuclear and functions as the nuclear transcriptional factor to induce multiple downstream effects. A series of recent studies show that AR could be activated without androgen binding which is considered as ligand independent activation. (Fig 1.2) In the adult prostate androgens promote survival of secretory epithelial cells, the primary step to malignant transformation to prostate adenocarcinoma (De Marzo, Nelson et al. 1998). Androgen-induced prostate epithelial cell proliferation is partially regulated by an indirect pathway involving paracrine mediators produced by stromal cells, such as insulin-like growth factor (IGF), fibroblast growth factor (FGF) and epidermal growth factor (EGF) (Cunha and Donjacour 1989; Byrne, Leung et al. 1996). The epidemiological evidence fails to establish a link between elevated serum testosterone, DHT, or adrenal androgens and prostate cancer risk, suggesting that androgens are not sufficient to promote prostate carcinogenesis (Roberts and Essenhigh 1986; Hsing 2001).

AR Interaction with Key Growth Factor Signaling Pathway EGF and its membrane receptor, the EGFR, play a vital role in the pathogenesis of different tumors, including prostate cancer (Russell, Bennett et al. 1998). Both the ligand and its signaling receptor partner are frequently up-regulated in advanced stages of prostate cancer (Di Lorenzo, Tortora et al. 2002). Inhibition of EGFR with monoclonal antibodies or with tyrosine kinase inhibitors suprewwes either growth or invasion of androgen-dependent and -independent prostate cancer cells in vitro (Bonaccorsi, Marchiani et al. 2004; Festuccia, Muzi et al. 2005). EGFR involves in proliferation and invasion of cancer cells (Wells, Kassis et al. 2002), and also participates in the formation of the plasma membrane structures (lamellipodia) that mediate migration through the basal membrane (Rabinovitz, Gipson et al. 2001). Increased EGFR expression enhances tumour invasiveness of mammary adenocarcinomas by increasing cell motility in vivo without affecting tumor growth (Xue, Wyckoff et al. 2006), pointing the key role exerted by the EGF/EGFR system in invasion and metastasis. Strong evidence on the interaction

between EGF/EGFR and androgen signaling, provides proof-of-principle that engagement of multi-crossed signals is crucial to the acquisition and maintenance of androgen sensitivity (Leotoing, Manin et al. 2007). Prostate specific antigen, (PSA), the downstream gene of the androgen regulatory axis was found to be induced by the administration of IL-6, which activates EGFR. (Hobisch, Eder et al. 1998; Ueda, Mawji et al. 2002). This evidence initially pointed to the involvement of EGFR in dictating AR outcomes in prostate cancer cells. ErbB2, a lead member of the epidermal growth factor receptor family of receptor tyrosine kinases, was shown to be overexpressed in prostate cancer during progression to androgen-independent metastatic disease. (Heinlein and Chang 2004). Elegant studies by independent investigators provide the mechanistic basis for this important correlative cross-talk between AR and Erb2: both groups demonstrated that modulation of AR signaling activity by the HER-2/new tyrosine kinase promotes androgen -independent prostate tumor growth in vitro and in vivo (Craft, Shostak et al. 1999; Yeh, Lin et al. 1999). More recently acquired evidence further supports the signaling interaction by indicating that knocking down of ErbB2 by siRNA impaired prostate cancer cell growth via targeting AR activity (Mellinghoff, Vivanco et al. 2004). Taken together these lines of evidence converge to the recognition of the ErbB2 kinase activity being required for optimal transcriptional activity of AR in prostate cancer cells. (Mellinghoff, Vivanco et al. 2004; Liu, Majumder et al. 2005) Androgens can post-transcriptionally regulate mRNAs containing an AU-rich element in the 3' untranslated region, including EGF, by regulating the binding of endogenous HuR to the AU-rich 3′UTRs of EGF mRNA (Myers, Oelschlager et al. 1999; Torring, Dagnaes-Hansen et al. 2003). The fact that androgens differentially regulate the expression of ARE-binding proteins known to bind to these instability elements, supports another involvement of androgens in the posttranscriptional regulation of EGF (Simons and Toomre 2000; DiNitto, Cronin et al. 2003; Kuhajda 2006). In a paradoxical reverse- like fashion, EGF reduces AR expression and blocks androgen-dependent transcription in differentiated cells, while it activates the AR promoter (Culig, Hobisch et al. 1994). This close EGF-AR mechanistic encounter is fundamentally important contributor to prostate tumor progression, but one has to also consider that the AR transcriptional activity can be enhanced/regulated by other peptide growth factors (Orio, Terouanne et al. 2002).

The AR interacts with the MAPK/extracellular signaling-regulated kinase kinase- 1 (MEKK1) and the epidermal growth factor-1 receptor (Abreu-Martin, Chari et al. 1999; Bonaccorsi, Carloni et al. 2004), as shown in Figure 1. Androgen-activated AR activates mitogen-activated protein kinase (MAPK) (Peterziel, Mink et al. 1999) and in a “functional-symmetry”, EGF-activated MAPK signaling cascade interferes with AR function towards down-regulation of androgen responsiveness. MEK inhibitor reverse the EGF-mediated AR down-regulation in differentiated cells, thus suggesting the existence of an inverse correlation between EGF and androgen signaling in non-tumor epithelial cells (Leotoing, Manin et al. 2007). Additional key signal transducers in this dynamic, include transducer activator of transcription 3 (Stat3), which is most probably required for AR activation by IL-6 towards promoting metastatic progression of prostate cancer (Abdulghani, Gu et al. 2008). Increased levels of Stat3 have been shown to lead to Stat3- AR complex formation in response to EGF and interleukin-6 (IL-6) (as shown on Fig 1). Moreover, Stat3 increases the EGF induced transcriptional activation of AR, while androgen pre-treatment increases Stat3 levels in an IL-6 autocrine/paracrine dependent manner suggesting an intracellular feedback loop (Aaronson, Muller et al. 2007). AR expression can also affect clathrin-mediated endocytosis pathway of EGFR, which plays an essential role in the signaling integrity of the receptor. Thus there is rapidly growing evidence to highlight the significance of engaging active cross-signaling by prostate cancer cells towards determining their survival and response to the microenvironment (Bonaccorsi, Nosi et al. 2007). The active integration of AR and EGFR signaling within the lipid raft microdomains in target cells provides an intriguing topological-twist to this cross-talk. Thus considering that the serine-threonine kinase Akt1 is a convergence point of the two hormonal stimuli and AR is localized in lipid raft membranes where it is stabilized by androgens (Freeman, Cinar et al. 2007), one could easily argue that the newly found membrane “domain” harboring AR is responsible to the non-genomic signaling pathways elicited by AR. The emerging concept that Akt1 is sensitive to manipulations in cholesterol levels, gains direct support from biochemical analysis verifying that a subpopulation of Akt1 molecules resides within lipid raft microdomains (Bauer, Jenny et al. 2003; Zhuang, Kim et al. 2005). Distinct changes in phosphorylation state of Akt1 in

response to androgen occur quickly but temporally independent in the raft and non-raft compartment, implicating processing of dissimilar signals. Interestingly, EGF triggers Akt1 phosphorylation via more rapid kinetics than those induced by androgens; this was recently documented by elegant studies on the sensitivity of EGFR family proteins to disruptions in cholesterol synthesis and homeostasis, supporting the functional significance of EGF signal transduction through lipid rafts (Freeman, Cinar et al. 2007). Signaling by insulin-like growth factor 1 (IGF1), is of major mechanistic and biological significance (Burfeind, Chernicky et al. 1996; Pollak, Beamer et al. 1998; Wolk, Mantzoros et al. 1998; Nickerson, Chang et al. 2001). In a mechanistic scenario fostering AR reactivation in a low androgen environment (Grossmann, Huang et al. 2001), insulin resistance and hyperinsulinemia correlate with an elevated incidence of prostate cancer (Fan, Yanase et al. 2007). High IGF1 levels in the serum correlate with an increased risk of prostate cancer (Pollak, Beamer et al. 1998; Wolk, Mantzoros et al. 1998), whereas IGF1 enhances AR transactivation under very low or absent androgen levels (Culig, Hobisch et al. 1994; Orio, Terouanne et al. 2002) and promotes prostate cancer cell proliferation (Burfeind, Chernicky et al. 1996). Endogenous AR expression as well as AR transcriptional activity are known to be regulated by insulin via activation of the phosphatidylinositol 3-kinase (PI3K) transduction pathway (Manin, Veyssiere et al. 1992; Manin, Martinez et al. 2000; Manin, Baron et al. 2002). Foxo1, as a downstream molecule becomes phosphorylated and inactivated by PI3K/Akt kinase in response to IGF1 or insulin, and subsequently suppresses ligand-mediated AR transactivation (as shown on Fig1). Foxo1 is recruited by liganded AR to the chromatin of AR target gene promoters and interacts directly with the C terminus of AR in a ligand-dependent manner and disrupts ligand-induced AR subnuclear compartmentalization. By interfering with AR-DNA interactions, Foxo1 reduces androgen-induced AR target gene expressions, ultimately suppressing the growth of prostate cancer cells, while IGF1/insulin-PI3K/Akt-induced phosphorylation of Foxo1 ameliorated this tumor suppression (Fan, Yanase et al. 2007). Several studies support a “locally-controlled” positive feedback between IGF1 and AR signaling in prostate cancer cells. Liganded AR up-regulates IGF1 receptor expression in HepG2 and LNCaP cells, presumably resulting in higher IGF1 signaling

tension in prostate cancer cells. (Sung, Hsieh et al. 2007) Two androgen response elements (AREs) within the IGF-I upstream promoter, act in cis to activate IGF-I expression (Sung, Hsieh et al. 2007). Androgens also impose a tight control on IGF signaling via modulation of IGF binding proteins (IGFBPs) in prostate epithelial cells, while both androgens and IGF-I up-regulate IGFBP-5 mRNA in androgen-responsive human fibroblasts (Yoshizawa and Ogikubo 2006). IGFBP-5 initially binds IGFs with high affinity, principally by an N-terminal motif, and inhibits IGF activity by preventing IGF interaction with the type 1 receptor (Kalus, Zweckstetter et al. 1998). Taken together, this evidence supports a strong “higher-level” interaction between the AR and IGF signaling, via recruitment of direct pathways towards transcriptional regulation and protein post-translational changes, all critical to prostate cancer cell survival. Vascular endothelial growth factor (VEGF), originally known as vascular permeability factor, is a well-characterized proangiogenic proteins and the most prominent cytokine responsible for endothelial cell differentiation, migration, proliferation, tube formation, and vessel assembly (Fong, Rossant et al. 1995). The role of VEGF in prostate cancer development and progression has been extensively studies. The value of VEGF has been investigated not only as screening test for advanced disease, but also as a therapeutic target. At the molecular level, the "hypoxia-response" signaling system upregulates the expression of a network of effectors that increase the propensity of tumor cells for survival, even in this adverse environment (Anastasiadis, Bemis et al. 2003). VEGF expression is transcriptionally activated by hypoxia-inducible factor in response to changes in oxygen tension within the microenvironment (Delongchamps, Peyromaure et al. 2006). Androgen-stimulated growth of the glandular ventral prostate in adult castrated rats is preceded by increased epithelial VEGF synthesis, endothelial cell proliferation, vascular growth, and increased blood flow (Joseph, Nelson et al. 1997; Franck-Lissbrant, Haggstrom et al. 1998). This early work highlighted the central role of VEGF in androgen-mediated prostate vascularity, a role that was further supported by recent evidence on the ability of androgens to increase endothelial cell proliferation, vascular volume, and organ weight in the mouse ventral prostate (Lissbrant, Hammarsten et al. 2004). In prostate cancer, the effect of androgens on angiogenesis is mediated via their

ability to regulate VEGF through activation of HIF in androgen-sensitive tumors (Boddy, Fox et al. 2005). The significant correlation between HIF-1a and HIF-2a expression and with AR and VEGF expression (Boddy, Fox et al. 2005; Banham, Boddy et al. 2007) provides proof-of-principle for such a control system. More recent studies point to the upregulation of vascular endothelial growth factor-C (VEGF-C) after androgen withdrawal in prostate cancer cells, as a critical event (Rinaldo, Li et al. 2007). Androgen deprivation also activates the small GTPase, RalA, whose activation leads to VEGF-C upregulation, while VEGF-C increases the expression of the AR co-activator BAG-1L that facilitates AR transactivation (at low androgen levels). The mechanistic link here is the intracellular reactive oxygen species that under androgen-withdrawal conditions induce RalA activation and VEGF-C synthesis (Rinaldo, Li et al. 2007). FGF family provides a large membership with broad-spectrum of functions, including cell migration and/or differentiation, as well as angiogenesis (Ornitz and Itoh 2001). Alterations in FGFs production and/or FGF receptors expression play key roles in prostate tumor progression, particularly in androgen-independent tumors. The estrogen receptor (ER), in coordinated action with AR activation, regulates the synthesis of FGF-2 and FGF-7, while stromal ER may mediate the synthesis of stromally-derived growth factors thus contributing to the pathogenesis of benign prostatic hyperplasia. A direct androgenic regulation of the FGF pathway in the prostate is supported by ample evidence. For example, AR signaling can dictate dramatic changes in the expression pattern of FGFs in both prostate tumor epithelial cells and prostate stromal cells, primarily via changes in FGF1, FGF2, FGF8, and FGF10 (Saric and Shain 1998; Nakano, Fukabori et al. 1999; Rosini, Bonaccorsi et al. 2002). Via a positive feedback, AR is upregulated by paracrine FGF10 and synergizes with cell-autonomous activated AKT in prostate cancer cells (Memarzadeh, Xin et al. 2007). Moreover, in response to FGFs, AR facilitates FGF-induced survival of prostate cancer cells, possibly through Bcl-2 induction and down-regulation of AR, allowing the escape of selected clones from androgenic control (Rosini, Bonaccorsi et al. 2002; Gonzalez-Herrera, Prado-Lourenco et al. 2006).

AR and TGFβ: Death Links Life Partners Transforming growth factor-β (TGF-β) is a ubiquitous cytokine that plays a critical role in numerous pathways regulating cellular and tissue homeostasis. The TGFβ superfamily members regulate proliferation, growth arrest, differentiation, and apoptosis of prostatic stromal and epithelial cells as well as the formation of osteoblastic metastases. TGFβ is overexpressed in most prostate tumors and regulates diverse functions of stromal cells through both Smad-dependent and Smad-independent signaling pathways (Coffey, Shipley et al. 1986; Roberts, Sporn et al. 1986; Derynck and Zhang 2003; Zhu and Kyprianou 2005). Recently cofilin and prohibitin, two novel signaling effectors of TGF-β1, that serve as potential intracellular effectors of its apoptotic action was identified in human prostate cancer cells (Zhu, Fukada et al. 2006). Cancer cells become refractory to the growth inhibitory activity of TGFβ due to loss or mutation of transmembrane receptors or intracellular TGFβ signaling effectors during tumor initiation (Akhurst and Derynck 2001). During prostate tumor progression to metastatic disease, TGF-β1 ligand overexpression results in prooncogenic rather than growth suppressive effect. In human prostate cancer cells TGFβ signaling proceeds via ligand binding and subsequent phosphorylation of receptor type II to the TβRI kinase to Smad activation (Zhu and Kyprianou 2005). Interaction of Smad4, (alone or together with Smad3), with the AR in the DNA-binding and ligand-binding domains, may result in the modulation of DHT induced AR transactivation (Zhu, Partin et al. 2008). Interestingly, in the human prostate cancer cell lines PC3 and LNCaP, addition of Smad3 enhances AR transactivation, while co-transfection of Smad3 and Smad4 actually repress AR transactivation (Kang, Huang et al. 2002). A protein-protein interaction between AR and Smad3 has been documented both in vitro and in vivo via the transcription activation domain of AR and the MH2 of Smad3; AR repression by Smad3 is mediated through the MH2 domain (Hayes, Zarnegar et al. 2001). In PC3 prostate cancer cells, AR expression reduces the TGFβ1/Smad transcriptional activity and the growth effects of TGFβ1 (in the absence of DHT) thus preventing TGFβ1 induced growth inhibition and apoptosis. Furthermore TGFβ1 suppresses the E2F transcriptional activity of AR activation by DHT, an event that is associated with a reduced c-Myc expression. A putative androgen response sequence

identified in TGFβ promoter may provide a mechanistic basis for TGFβ promoter activity towards DHT in a dose-dependent manner in both Huh7 and PC3/AR expressing cells. The AR mediated upregulation of TGFβ1 demonstrated in human HCC xenografts derived from AR-overexpressed Huh7-cells, implicates AR functional interaction with TGF-β1 in hepatocarcinogenesis by (Yoon, Kim et al. 2006). In LNCaP cells either with or without overexpression of TGFβ receptors (Tβ RI and TβRII), androgens down-regulate TGF-β1-induced expression of TGF-β1, c-Fos, and Egr-1, while DHT inhibits TGF-β1 transcription (Chipuk, Cornelius et al. 2002).The AR- associated protein 55 (ARA55/Hic-5) (which belongs to the LIM protein superfamily) might be a critical regulator in this AR-TGF-β1 crosstalk. Such a role is supported by evidence that overexpression of ARA55 inhibits TGFβ-mediated up-regulation of Smad transcriptional activity in NRP-154 and NRP-152 rat prostate and LNCaP human prostate cells. In addition, interaction between ARA55 and Smad3 occurs through the MH2 domain of Smad3 and the C terminus of ARA55 (Wang, Song et al. 2005). The involvement of AR in the apoptosis outcomes of TGFβ signaling in prostate cancer cells has been established by work from our group. Treatment of TGFβ receptor II overexpressing LNCaP TβRII cells with TGFβ in the presence of DHT, both cell cycle arrest and apoptosis induction are significantly enhanced over TGFβ alone, through caspase-1 activation and targeting of bcl-2 (Bruckheimer and Kyprianou 2001). Enforced bcl-2 expression significantly inhibits the combined TGFβ and DHT apoptotic effect in prostate cancer cells (Bruckheimer and Kyprianou 2002). An androgenic contribution, with TGFβ enhancement, on the epithelial-mesenchymal transition (EMT) provides an attractive mechanistic possibility in view of the assigned role of EMT during cancer metastasis (Zavadil and Bottinger 2005), with E-cadherin being considered as a potential target for such a dynamic duo.

AR Plays in the Stroma: Functional Promiscuity In the prostate microenvironment the stroma, is a leading component of the tumor dynamics. Stroma-derived fibroblasts play an active role in carcinogenesis in addition to structurally supporting the epithelial cell growth (Chung, Chang et al. 1989; Camps,

Chang et al. 1990; Chung, Gleave et al. 1991; Cunha, Hayward et al. 1996). Elegant studies in the early 1990s established that human prostate-derived stromal cells stimulate growth of prostate cancer cells in vitro and in vivo (Gleave, Hsieh et al. 1991). This evidence widely popularized the belief that disturbance in the epithelial–stromal interactions is most critical in the pathogenesis of prostate cancer (Hayward, Grossfeld et al. 1998). Androgenic control during normal growth and differentiation of the prostate gland is regulated via nuclear AR in both stomal and epithelial cells (Sar, Lubahn et al. 1990). The close association between low AR levels in the stroma adjacent to malignant epithelium, with a poor clinical outcome in prostate cancer patients attracts major translational significance (Henshall, Quinn et al. 2001). Androgens increase VEGF transcription and secretion of biologically active VEGF from human prostatic stroma, thus indirectly enhancing prostate cancer growth and angiogenesis (Levine, Liu et al. 1998). DHT and FGF can synergistically stimulate prostate stromal cell proliferation (Niu, Xu et al. 2001), while castration-induced androgen depletion rapidly reduces stroma IGF-1 synthesis and action in the prostate epithelium. The normally “closed-circuit” rules of compartmentalization become “loose” here: although IGF-1 is principally produced in the stroma and IGF-R1 in the epithelium, both are under androgenic regulation as stroma IGF-1 mRNA is significantly decreased after castration, correlating with epithelial cell apoptotic response (Ohlson, Bergh et al. 2007). TGF-β1 is a also regulator of stromal cell proliferation and differentiation, depending on the specific stromal cell type, microenvironment, and contributing activities of other growth factors (Sporn and Roberts 1992). A distinct in its complexity cross-talk between androgens and TGF-β1 signaling in prostate stromal cells, affects AR localization, cell proliferation, and myodifferentiation, thus defining its mechanistic contribution to the reactive stroma that drives prostatic tumors. Indeed AR expression and TGF-β1 levels significantly correlate in the stromal tissue of prostatic intraepithelial neoplasia (PIN) (Cardillo, Petrangeli et al. 2000). While androgens induce rat PS-1 prostate stromal cell proliferation and specific gene expression, TGF-β1 can directly antagonize these androgen-dependent effects on prostate stroma. Finding a good “platform” in the prostate stroma, TGF- β1 triggers a cytoplasmic translocation of nuclear AR in PS-1 cells, thus defining another niche for interaction with the androgen axis

(Gerdes, Dang et al. 1998). TGF-β1 results in an AR nuclear-cytoplasmic-nuclear translocation during myodifferentiation (Gerdes, Larsen et al. 2004), while androgens increase proliferation of prostatic smooth muscle cell PSMC1 via TGFβ1 secretion (Salm, Koikawa et al. 2000).

Summary During prostate cancer progression the androgen axis engages the growth factor network to an active cross-talk towards maintaining the abnormal biological status of prostate cancer cells. This intimate signaling interaction between the AR and growth factors pathways provides an attractive platform for dissecting the molecular mechanism underlying the emergence of hormone-refractory prostate cancer. Androgens can indeed change the outcomes of growth factor signals from growth-inhibitory to tumor promoting in advanced prostate cancer, and the impact of such crosstalk on the metastatic process opens new directions regarding the fate of the cell. Opportunities for therapeutic intervention may also emerge from further ‘analytical’ dissection of the crosstalk between AR and the growth factor network/their signaling effectors, not only in prostate cancer epithelial cells but also in the context of the tumor microenvironment.

Figure 1. 1 The Anatomy of the Prostate Gland

(Adopted from www.wikipedia.com). The prostate is part of a man's reproductive system. It's an organ located in front of the rectum and under the bladder. The prostate surrounds the urethra, the tube through which urine flows. A healthy prostate is about the size of a walnut. If the prostate grows too large, it squeezes the urethra. This may slow or stop the flow of urine from the bladder to the penis.

Figure 1.1

Figure 1. 2 AR Signaling Pathway

The left part of the schema shows the classical AR signaling pathway. Androgens bind to AR and release AR from HSP90, the androgen and AR complex translocated to the nuclear and induces the downstream transcriptional activity. The right part shows the ligand-independent acitvaiton of AR: AR is phosphorylated with multiple cell signaling molecules, the phosphorulated AR is translocated to nuclear and induced the downstream effect. (Adopted the following article: Mechanisms of Disease: the role of heat-shock protein 90 in genitourinary malignancy Jean-Baptiste Lattouf, Ramaprasad Srinivasan, Peter A Pinto, W Marston Linehan and Leonard NeckersNature Clinical Practice Urology (2006) 3, 590-601)

Figure 1.2

Figure 1. 3 Growth Factors Crosstalk with AR in Prostate Cancer Cells.

IGF, FGF, VEGF and TGFβ secreted by the prostate stromal cells activate their receptors and interact with AR signal axis. Androgen signal involve in regulating of prostate cancer cells secreted VEGF and TGF which affect the microenvironment in the prostate cancer by inducing angiogenesis and stromal cell growth and differentiation. EGF signal encounter AR signal bypassing multiple pathways. The growth factors signals go through AR signal and regulate the downstream effectors of AR including proliferation, differentiation, apoptosis, survival etc. which are critical factors in the prostate carcinogenesis and cancer progression. (Zhu and Kyprianou, "Androgen receptor and growth factor signaling cross-talk in prostate cancer cells." Endocr Relat Cancer 15(4): 841-849.)

Figure 1.3

Table 2. 1 Cell lines

CHAPTER TWO EXPERIMENTAL DESIGN AND METHODS

Cell Lines The androgen-sensitive and TGFβ responsive human prostate cancer cells LNCaP TβRII cells (generated in our laboratory) (Guo and Kyprianou 1998; Guo and Kyprianou 1999), and the parental LNCaP, CWR22 and PC3 prostate cancer cell lines were used. The characters of these cell lines are listed in Table 2.1. The human breast cancer cells MCF-7 and the human renal cancer cells 786-0 were obtained from ATCC (Bethesda). To determine the effects of exogenous DHT (Sigma, St; Louis, MO) and TGFβ (R& D, Minneapolis, MN), cells were grown in DMEM or RPMI 1640 with 10% fetal bovine serum (FBS; without phenol red) and transferred to medium 5% charcoal strip serum (CSS) prior to treatment.

Antibodies and Chemicals Antibodies against E-cadherin, β-catenin, Parp were purchased from Cell Signaling Technology (Danvers, MA); antibodies against AR, tubulin, N-cadherin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); antibodies against the cofilin and actin were purchased from Sigma-Aldrich (St. Louis, MO); antibody against talin was purchased from Upstate Biotech (Billerica, MA); the antibody against GAPDH are purchased form Novus biologicals (Littleton, CO). Casodex was a generous gift from Dr. Chendil Damodaran, University of Kentucky College of Health Sciences. Paclitaxel and Nocodazole was purchased from Sigma-Aldrich (St. Louis, MO); Velcade and TRAIL (TNF Related Apoptosis Inducing Ligand) were generous gift from Dr Steven Schwarze (Department of Molecular Biochemistry, University of Kentucky College of Medicine).

Transfections Subconfluent cultures of PC3 or LNCaP cells, were transfected with the pCDNA-Zeo AR vector or AR shRNA vector (Open biosystem, Huntsville, AL), using the Lipofectamine 2000 Reagent (Invitrogen; Carlsbad, CA, USA). pCDNA-zeo AR construct was prepared by cloning full AR fragment from pCMV5-AR vector (BamHI/XhoI). pCMV5-AR was a generous gift from Dr. Donald Tindall (Mayo Clinic).

After transfection (exposure to plasmid DNA for 6hrs at 37°C, 5% CO2), the growth medium was changed to 10% FCS for 48hrs, prior to selection in antibiotic containing medium (25μg/ml zeosin/puromycin) (Invitrogen; Grand Island, NY, USA). Individual colonies were selected, cloned and grown in 10% FCS containing medium. Protein expression of transfected AR was examined by Western blotting.

Wounding Assay Cells cultures (80% confluency) were subjected to wounding as previously described (Tahmatzopoulos, Sheng et al. 2005). The number of cells migrating to the wounding area was counted at the end points indicated.

Flow Cytometry Analysis Prostate cancer cells (1x106) were labeled using a specific primary antibody and stained by fluorescent-conjugated secondary antibody. Samples were subjected to fluorescence analysis using the PARTEC System (Münster, Germany). Cellular DNA content was measured by cell cycle analysis. Cells were collected, washed, fixed in 70% ethanol and incubated with RNase (Sigma–Aldrich). After fixation, the cells were washed with PBS and stained with the DNA fluorochrome propidium iodide (50 μg/mL, Sigma– Aldrich) for 15 min at room temperature. Propidium iodide fluorescence was measured by flow cytometry (FACScan, BD Biosciences, USA). A minimum of 20,000 cells were acquired per sample. The percentage of cells in G0/G1, S and G2/M was determined from DNA content histograms.

Western Blot Analyses Total cellular protein was extracted from the cell pellets by homogenization in RIPA buffer (50mM Tris; 150 mM Nacl; 0.1% SDS; 0.5% Na.Deoxycholate; 1% TritonX-100 or NP40; 1mM PMSF). Protein samples (20-60μg) were loaded on 4%/12% SDS- polyacrylamide gels and subjected to electrophoretic analysis and subsequent blocking. Membranes were incubated with the primary antibody (overnight at 4oC) and the relevant secondary antibodies (1hr at room temperature). The E-cadherin, β-catenin, vimentin and Parp antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA); The AR, tubulin, N-cadherin antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); The cofilin and α-actin antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA); The antibody against talin1 was a generous gift from Dr. R. McCann (Mercer College, GA, USA); The GAPDH antibody was purchased from Novus Biologicals (Littleton, CO, USA).

Invasion Assay

The invasion ability of prostate cancer cells was determined using the transwell chamber assay. Matrigel (1mg/ml) in serum free-cold cell culture media was placed in the upper chamber of a 24-well transwell and incubated for 5 hrs at 370C. Cells were harvested and cell suspensions (100μl) were placed on the matrigel and the lower chamber of the transwell was filled with culture media in the presence of 5μg/ml fibronectin, as an adhesive substrate. DHT (1nM) was added in both upper and lower level chambers. Following 48hr incubation at 370C, transwells were removed and stained with Gimsa solution. Non-invading cells on top of transwells were removed and invading cells were counted under the microscope.

Immunofluorescence Staining Cells were plated (1x105cells/well) in chamber slides and after 24hrs cells were incubated with RPMI 1640 + 10% CSS supplemented with either DHT (1nM), TGFβ (5ng/ml), or the combination of DHT (1nM)-TGFβ (5ng/ml), as indicated. Following

treatment, cells were fixed in 2% paraformaldehyde in phosphate-buffered saline (PBS) and permeabilized in 0.1% Triton X-100 in PBS. Cells were stained by incubation with the primary antibody (overnight at 4oC), followed by exposure to the secondary immunofluorescence antibody and FITC-phalloidin, (1hr at room temperature). FITC- phalloidin was purchased from Sigma-Aldrich (St. Louis, MO, USA); Slides were mounted by Vectashield mounting medium (Vector laboratories Inc, Burlingame, CA, USA).

RNA Extraction and Realtime RT-PCR RNA samples extracted with Trizol Reagent were treated with RNase-free DNase

I and reversetrancript to cDNA (Biorad, Hercules, CA) Taqman realtime RT-PCR analysis of the cDNA samples was conducted in an ABI 7700 Sequence Detection System (Applied Biosystems Inc, Foster City, CA) with the specific primers of E- cadherin and Snail (Applied Biosystems Inc, Foster City, CA).

Patient Population Between January 2001 and November 2004, 57 patients with high-risk localized prostate cancer (defined a cT2b or T3a or PSA > 15 ng/ml or Gleason grade > 4+3) were recruited for a phase II trial clinical trial of neoadjuvant chemotherapy (using docetaxel and mitoxantrone). The design of the clinical trial has been previously described (Beer, Garzotto et al. 2004). The study was approved by the Institutional Review Boards of the Oregon Health & Science University, Portland VA Medical Center, Kaiser Permanente Northwest Region, Legacy Health System, and the University of Washington and all patients provided signed informed consent.

Specimen Collection and Specimen Processing The specimen collection and processing were performed by our collaborator in Oregon Health and Science University. From each patient, ten standard prostate biopsies

(bilateral at the apex, bilateral medial and lateral at mid-gland, bilateral medial and lateral at the base of the gland) were obtained under ultrasound guidance and snap-frozen in liquid nitrogen. Biopsy material from twenty representative patients was used for gene expression profiling. Frozen sections (7 µM) were cut from biopsy tissue frozen in OCT blocks, stained with Mayer’s hematoxylin (Sigma, St. Louis, MO), dehydrated in 100% ethanol and xylene, and used for laser-capture microdissection (LCM) using an Arcturus PixCell II microscope (Arcturus, Mountain View, CA).

Tissue Microarray Construction A tissue microarray (TMA) was constructed from formalin fixed representative tissues collected at prostatectomy from the first 50 patients enrolled on the neoadjuvant chemotherapy study. Tissue cores (0.6 mm diameter placed 0.2 mm apart) were removed from the paraffin-embedded prostate tissue blocks (donor blocks) and placed in a recipient paraffin block (30 x 25 mm) using a precision Tissue Arrayer (Beecher Instruments, Sun Prairie, WI). H&E slides of each donor block were examined microscopically and reviewed by a pathologist to determine the appropriate location. From every patient, three cores each of prostate cancer, normal prostate, and, where applicable, lymph nodes with metastatic cancer were placed in each block in a pseudo- randomized fashion. Dispersed amongst the study cores were control tissues from non- study patients (liver, prostate, lymph node, salivary gland, kidney, testis), untreated cell lines (DU-145, PC3, LNCaP), and the same cell lines treated with mitoxantrone and docetaxel (singly and in combination). After completion, the block was heated in a 37ºC oven for 30 minutes to ensure incorporation of the cores into the block. The block was then cut into 5µm thick sections and the unstained slides were stored at 4°C until needed for staining. Microscopic evaluation of frozen sections of tissue samples identified the presence of adequate number of cancer cells in both pre-treatment and post-treatment samples for 31 subjects. Frozen sections (7 Amol/L) were cut from tissue frozen in ornithine carbamyl transferase blocks, stained with Mayer’s hematoxylin (Sigma), dehydrated in 100% ethanol and xylene, and used for laser capture microdissection using an Arcturus PixCell

IIe microscope (Arcturus, Inc.). To evaluate gene expression alterations after chemotherapy, neoplastic epithelium from pretreated biopsy and posttreated prostatectomy specimens were captured separately (3,000 cells per sample). The histology of captured cells was verified both by review of an H&E-stained frozen section from each sample and by review of the pre/post–laser capture microdissection images.

Immunocytochemical Staining PSA and AR expression in the tumor epithelial was assessed by using a standard immunoperoxidase method (Dakocytomation LSAB2 System-HPR, Carpinteria, CA) Citrate buffer and proteinase K solution (20 Ag/mL) was used for antigen retrieval. Serial sections were exposed to monoclonal anti-PSA IgG antibodies and the rabbit polyclonal antibody against E-cadherin from Cell Signaling Technology Inc. (Danvers, MA); N- cadherin, and AR (# sc-7939, and sc815 respectively, Santa Cruz Biotechnology, Santa Cruz, CA). Overnight at 4OC (negative controls consisted of incubation with rabbit- polycolone IgG antibody). Sections were subsequently exposed to biotinylated goat anti- rabbit IgG and horseradish peroxidase – streptavidin conjugate (Chemicon). Color development was accomplished using a FAST 3,3-diaminobenzidine based kit (Sigma- Aldrich), and counterstained with hematoxylin. Images were captured using an Olympus BX51 microscope system (Olympus America). Protein expression and localization were assessed in formalin-fixed, paraffin-embedded prostate cancer TMAs via light microscopic examination, while blinded to treatment modality. The overall pattern of staining in human prostate tumor cells in the TMAs was determined as the average number of positive epithelial cells in three different fields for each tissue core.

Immunoprecipitation Analysis Cell were lysed in lysis buffer [10 mM Tris pH 7.5, 1 mM EDTA, 400 mM NaCl, 10% glycerol, 5 mM NaF, 0.5 mM sodium orthovanadate, 1 mM dithiothreitol (DTT), and Complete Mini Protease Inhibitor Cocktail (Roche Biochemicals, Indianapolis, IN)]. Cell extracts were homogenized and protein content was quantitated using the Biorad

Protein Assay (Biorad, Hercules, CA). Lysates (400 mg) were precleared with protein A/G beads (Oncogene Research Products, #IP05, Boston, MA) and precleared lysates were incubated with AR or a-tubulin antibody overnight at 4C. 10ul protein A/G beads were subsequently added to the cell lysate/ antibody mixture. Following incubation (1 hr at 4C) the lysate/antibody/bead mixture was centrifuged at 14,000g (30 sec). Following 3 times washes with PBS, the beads were subjected to elution with 100 mM glycine pH 3.0; eluate-fractions were centrifuged (2 min at 14,000g) in 1M phosphate buffer pH8.0 and final samples were lysed in SDS–PAGE lysis buffer and subjected to Western blotting.

Luciferase Activity Assays Cells were plated (105cells/well) in six-well plates and treated as described above. After 48 hsr, cells were transfected with 1 mg/well ARE luciferase construct (from Dr. Zoran Culig, Inssbruck, Austria) in the presence of the control Renilla luciferase construct (Promega, Madison, WI) using Tfx-50 transfection reagent (Promega, # E1811). Following a 2 hr-incubation with the DNA/Tfx50 mixture, serum-containing media (2 ml) were added to the cells and incubation was continued for an additional 22 hr. After treatment cells were harvested and luciferase activity was determined according to the manufacturer’s protocol (Promega, Dual Luciferase Assay, # E1920).

Statistical Analysis One-way analysis of variance (ANOVA) was performed using the StatView statistical program to determine the statistical significance between values. All numerical data are presented as mean values ± SEM (standard error of the mean). A p value less than 0.05 was considered statistically significant.

CHAPTER THREE

ANDROGREN RECEPTOR CROSSTALK WITH TGFβ SIGNALING PATHWAY

Introduction Dissection of the cross-talk between key growth factor signaling and the androgen/ AR axis will enable a better understanding of the mechanisms underlying apoptosis deregulation in hormone-refractory prostatic tumors, leading to the identification of new cellular targets for therapeutic intervention in patients with advanced disease (Bruckheimer and Kyprianou 2001). Growth factors also contribute to the development of androgen-independent phenotype via autocrine production of growth stimulatory factors, or by altered responsiveness to growth inhibitory and apoptotic factors (Bottinger, Jakubczak et al. 1997; Guo, Jacobs et al. 1997). TGF-β serves as an autocrine growth inhibitory factor in the normal and malignant prostate (Kyprianou and Isaacs 1989; Tang, de Castro et al. 1999; Shariat, Menesses-Diaz et al. 2004). TGF-β elicits its effects by inducing a heteromeric complex formation of two types of transmembrane receptors type I and type II serine threonine kinases.(Massague and Gomis 2006). As a consequence, the TβRII kinase phosporylates TβRI, thereby activating its serine-threonine kinases (Wrana, Carcamo et al. 1992; Wrana, Attisano et al. 1994; Derynck, Akhurst et al. 2001). The intracellular domains of these receptors activated by the ligand binding, control the recruitment and activation of the intracellular effectors, the Smads. The two cytosolic Smads, Smad2 and Smad3 first become transiently associated with and phospohorylated by the TβR-I kinases (Tsuchida, Lewis et al. 1993; Massague 1998; Derynck and Zhang 2003). Activation of the pathway- specific Smads results in a complex formation with Smad4 and upon nuclear translocation this complex functions as a transcriptional regulator of target gene expression (Derynck and Zhang 2003). Both Smad2 and Smad4 are mutated in a significant proportion of colorectal and pancreatic tumors and less frequently in breast and lung cancers; however mutations in either of these TGF-β signaling effectors are

infrequent in prostate cancer (Guo, Jacobs et al. 1997). Receptor-activated Smads are linked to the transcriptional machinery through a direct physical interaction with the transcriptional coactivator CBP/p300 (Massague 1998). Upon nuclear translocation, Smad4 complexes activate specific target genes through cooperative interactions with DNA and DNA-binding proteins such as Fos/Jun (Derynck, Zhang et al. 1998; Zhang, Feng et al. 1998; Feng and Derynck 2005). Insensitivity to TGFβ due to a dysfunctional signaling, contributes to the early stages of tumorigenesis due to loss of apoptosis control (Roberts, Anzano et al. 1985; Tang, de Castro et al. 1999) This chapter focuses on the cross-talk between TGFβ and androgens and the importance of AR integrity in apoptosis outcomes in human prostate cancer cells, demonstrates that DHT enhances TGF-β-mediated apoptosis of prostate cancer cells via the interaction of AR with Smad4 in the LNCaP TβRII cells. In the PC3 prostate cancer cells (TGFβ responsive) however, the presence/overexpression of wtAR, not only fails to mediate the apoptosis promoting effect of DHT, but also abrogates apoptosis induction by TGF-β, suggesting a differential effect of the wt AR in TGFβ mediated apoptosis and transcriptional activity.

TGFβ Regulates AR-dependent Gene Expression in Prostate Cancer Cells To gain an insight into the dynamics of a potential cross-talk between TGFβ and androgens we first investigated whether TGFβ operates in an AR-dependent transcription we examined the combined effect of TGFβ and DHT on an androgen- responsive element, the probasin promoter activity in LNCaP-TβRII cells. LNCaP-Tβ RII cells were transfected with the probasin luciferase construct and Renilla luciferase construct and treated with 1nM DHT and/or TGFβ (5.0 ng/ml) for 24 hrs. The data shown on Figure 2.1A indicates that TGFβ enhances probasin luciferase activity. TGFβ also synergistcally increases DHT-induced probasin promoter activation. The effect of DHT and/or TGFβ on androgen-regulated genes, was assessed by examining PSA expression in the androgen-sensitive and responsive human prostate cancer LNCaP TβRII. PSA protein expression was evaluated after treatment by Western blot analysis. As shown on Figure 2.1B marked increase in PSA expression levels was detected after two days of

TGFβ treatment. A further significant increase in PSA expression was observed in response to DHT and TGFβ combination treatment (Fig 3.1B)

Impact of TGFβ and DHT Interaction on TGFβ mediated Transcription To determine the players of a TGFβ mediated transcriptional regulation during the apoptotic response, LNCaP-Tβ RII cells were transfected with the WWP-luc p21 luciferase promoter construct and Renilla luciferase construct and treated with DHT and/or TGFβ for 24hrs. The results summarized on Figure 3.2A indicate that DHT alone had no significant effect on p21 promoter activity in LNCaP-Tβ RII cells. However, DHT could increase TGFβ induced p21-promoter activation. It was previously demonstrated that DHT promotes TGFβ induced apoptosis in LNCaP-TβRII cells.(Bruckheimer and Kyprianou 2001) To investigate the functional requirement of DHT in TGFβ induced apoptosis, we evaluated the apoptotic response of the LNCaP-TβRII cells to TGFβ and/DHT in the presence or absence of an anti-androgen (Casodex) and TGFβ neutralizing antibody. As shown on Figure 3.2B, the combination treatment of DHT and TGFβ of LNCaP-TβRII cells in the presence of Casodex, led to a significant decrease in apoptosis. Targeting the TGFβ signaling by the TGFβ neutralizing antibody also resulted in a marked reduction in the apoptotic response to androgen/TGFβ (Fig. 3C).

TGFβ Increases AR Nuclear Translocation Activity AR nuclear translocation is a critical event for AR transcriptional activity. To study the ability of TGFβ to induce the AR nuclear translocation, LNCaP-TβRII cells were treated with DHT (1nM) and/or TGFβ (5.0ng/ml) for 6, 24 and 48hrs. Immunofluorescence AR staining revealed that AR was localized in both the cytoplasm and nuclear in the untreated LNCaP-TβRII cells. As shown on Figure 3.3, there was a significant AR nuclear translocation after 24-48hrs treatment with DHT; Exposure to TGFβ alone also resulted in nuclear AR translocation within 24hrs. In response to the DHT /TGFβ combination, an earlier AR translocation to the nucleus was detected, i.e within 6hrs of treatment (Fig. 3.3).

DHT and TGFβ Promote AR-Smad4 Association To gain insight into the mechanism via which TGFβ represses transcription of AR-dependent promoters, we used an immunprecipitation approach to investigate whether there was a physical interaction between AR and the intracellular effectors of TGFβ, the Smad proteins. Figure 3.4 indicates a representative immunoprecipitation analysis revealing the AR-Smad4 complex formation in response to the combined treatment of DHT/TGFβ. The results demonstrate that DHT promotes the interaction between AR and Smad 4 in both the LNCaP-TβRII cells and the PC3 AR prostate cancer cells (Fig. 3.4). A similar response was obtained in response to TGFβ (3 days).

Apoptosis Outcomes in Response to TGFβ and DHT in PC3 Prostate Cancer Cells Expressing Wild-type AR To determine whether the apoptosis-promoting effect by DHT in prostate cancer cells was dependent on the status and integrity of AR, i.e. the wild type AR vs. mutant AR cross-talk with TGFβ1 signaling effectors, the wild type AR gene (T7 AR plasmid) was overexpressed in the human androgen-independent (AR lacking) but TGFβ1 responsive prostate cancer cells PC3. Stable transfectants thus generated, were treated with DHT and/or TGFβ and their response was analyzed. Apoptosis and cell proliferation were examined in PC3-neo and PC3-AR stable transfectants cells. DHT failed to enhance TGF-β1 mediated apoptosis in PC3 wt AR cells (Fig. 3.5A). Moreover wtAR overexpression in these PC3 cells abrogated TGF-β1 apoptosis compared to the PC3 neo transfectants (Fig. 3.5A). Inversely, TGFβ did not enhance DHT induced proliferation in these cells (Fig. 3.5B), as in LNCaP-TβRII cells (mt AR).

The ability of TGFβ to affect AR regulated transcriptional activity was subsequently examined; PC3-neo and PC3-AR cells were transfected with the ARE luciferase promoter construct and Renilla luciferase construct and treated with DHT and/or TGFβ for 24 hrs. As shown in Figure 3.5C in PC3-neo control cells, the DHT and TGFβ combination failed to elicit an ARE activation; In contrast in PC3 cells wt AR-

transfectants (Clone 10), there was a robust induction of ARE transcription activity in response to DHT, as well as the DHT/TGFβ combination treatment. Furthermore TGFβ alone only modestly induced ARE transcriptional activity in prostate cancer cells with wt AR (Fig. 3.5C).

Figure 3. 1 Effect of TGFβ and DHT on Probasin Luciferase Activity and PSA Expression in LNCaP TβRII Cells.

A) Effect of TGFβ and DHT on probasin luciferase activity in LNCaP TβRII cells. LNCaP TbRII cells were transfected with the probasin luciferase construct and Renilla luciferase construct and treated with 1nM DHT and/or 5.0 ng/ml TGFβ for 24 hrs. Cells were harvested and luciferase activity was measured as described in “Materials and Methods”. The numerical data shown represent the average values from three independent experiments performed in duplicate. Statistical significance *, p<0.005. B) Effect of TGFβ and DHT on prostate specific antigen (PSA) protein expression in TGFβ sensitive LNCaP cells. LNCaP TβRII cells were treated with DHT (1nM) and/or TGFβ (5.0 ng/ml) for 1, 2, and 3 days. Cells were harvested and lysed in RIPA buffer. Protein samples (40 ug) were subjected to Western blot analysis in (12%) SDS-PAGE gels, transferred to nitrocellulose, and probed with the PSA antibody.

Figure 3.1

Figure 3. 2 Regulation of TGFβ Transcriptional and Apoptotic Activity by Androgens.

A) Effect of TGFβ and DHT on p21 Promoter Activity in LNCaP T β RII Cells. LNCaP T β RII cells were transfected with the WWP-luc p21 luciferase promoter construct and Renilla luciferase construct and treated with DHT (1nM) and/or TGFβ (5.0 ng/ml) for 24hrs. Following treatment, the cells were harvested and luciferase activity measured as described in “Materials and Methods”. Data represent the average values from three independent experiments performed in duplicate. * p value< 0.005. B and C): Effect of an antiandrogen and TGFβ neutralizing antibody on DHT and TGF-β-mediated apoptosis in LNCaP TβRII cells respectively. LNCaP TβRII were treated with DHT and/or TGFβ for 3 days in the presence of Casodex (5uM) (B) or a neutralizing anti-TGFβ antibody (10mg/ml) (C). Following treatment, the cells were fixed and stained with Hoechst 33342. The fragmented nuclei of apoptotic cells were observed by fluorescence microscopy (UV filter) and quantitated. Values represent the mean from two independent experiments performed in duplicate.

Figure 3.2

Figure 3. 3 Effect of TGFβ and DHT on AR Nuclear Translocation in LNCaP-TβRII Cells.

LNCaP-TRII cells were treated with DHT and/or TGFβ for 6-48 hrs and were LNCaP- TRII cells were treated with DHT and/or TGFβ for 6-48 hrs and were subsequently fixed. After exposure to the AR specific antibody, followed by fluorescein labeled secondary antibody (A), cells were visualized under fluorescence microscopy. The ratio of cell nuclear translocation for each treatment was evaluated (B). The mean value is determined by three independent experiments. *, p<0.05

Figure 3.3

Figure 3. 4 Combination of DHT and TGFβ Results in AR-Smad4 Association in Prostate Cancer Cells.

LNCaP-TβRII (A) and PC3-AR (B) transfectant cells were treated with DHT (1nM) and/or TGFβ (5ng/mL) for 6hrs. Cell lysates were prepared and subjected to immunoprecipitation analysis. Elutes of protein A/G beads was subjected to electrophoretic separation on 6% SDS-PAGE gels, transferred to nitrocellulose, and probed with antibodies specific to Smad 4 or AR.

Figure 3.4

Figure 3. 5 Effect of TGFβ and DHT in PC3-AR Cells.

PC3-neo and PC3-T7AR cells were treated with DHT (1nM) and/or TGFβ (5ng/ml); after 24hrs of treatment apoptosis was detected by Hoechst staining. The percentage of cells exhibiting apoptotic morphology was comparatively analyzed in the two groups (A). Cell proliferation was determined by [3H]-thymidine incorporation assay. (B) PC3-neo and PC3-T7AR cells were transfected with the ARE luciferase construct and Renilla luciferase construct and treated with DHT and/or TGFβ for 24hrs. Cells were subsequently harvested and luciferase activity was measured as described above. The numerical data represent the average values from three independent experiments performed in duplicate. *, p<0.005(C)

Figure 3.5

CHAPTER FOUR

EFFECT OF ANDROGEN AND ANDROGEN RECEPTOR ON EPITHELIAL- MESENCHYMAL TRANSITION (EMT) IN PROSTATE CANCER CELLS

Introduction Androgen action proceeds via an axis involving testicular synthesis of testosterone, its transport to target tissues, and its conversion by 5 -reductase to the active metabolite 5 -dihydrotestosterone (DHT). Androgens exert their biological effects by binding to the androgen receptor (AR) and inducing its transcriptional activity. The 5α-reductase enzyme is present in the urogenital sinus before and during prostate development (Siiteri and Wilson 1974; Heinlein and Chang 2002), and its inhibition during fetal development results in partial prostate development (Imperato-McGinley, Binienda et al. 1985). In adult males, androgens promote secretory epithelial cell survival, the cells primarily undergoing transformation in prostate adenocarcinoma (De Marzo, Nelson et al. 1998). Androgen deprivation is the only clinically effective therapy for advanced prostate cancer; however, due to relapse of castration-resistant androgen- independent tumors, the long-term benefit of androgen deprivation in patients with metastatic disease has been debated (Makhsida, Shah et al. 2005; Shahinian, Kuo et al. 2005; Lu-Yao, Albertsen et al. 2008).

The process of epithelial-mesenchymal transition (EMT), is a critical event during embryonic development, required for morphogenetic movements during parietal endoderm formation, gastrulation, as well as formation of organs and tissues (e.g. neural crest, heart, craniofacial structures) (Thiery 2003). A growing body of recent evidence links EMT to tumor progression and metastasis. Loss of epithelial-cell markers (e.g. E- cadherin, β-catenin) and gain of mesenchymal-cell markers (e.g. N-cadherin, Vementin), particularly at the leading edge or invasive front of solid tumors, has been reported in human tumor specimens and is associated with tumor progression to metastasis (Thiery 2002). Epithelial tumor cells lose cell polarity and cell-junction proteins and at the same time acquire protein mesenchymal-cell markers (e.g. N-cadherin, vimentin) and signaling activities associated with mesenchymal cells facilitating migration and survival in an

anchorage-independent environment and ultimately metastasis (Thiery 2002; Huber, Kraut et al. 2005). Pathological EMT in tumor cells results from transcriptional reprogramming of abnormal survival signals via receptors such as platelet derived growth factor receptor (PDGFR); fibroblast growth factor receptor (FGFR); transform growth factor-β receptor (TGFβR); insulin-like growth factor-1 receptor (IGF-1R); and regulatory kinases such as PI3K, AKT and mTOR (Xie, Law et al. 2004; Thiery and Sleeman 2006). TGFβ is a potent EMT inducer in normal development and organ homeostasis, as well as during tumor progression (Derynck and Akhurst 2007). TGFβ induces EMT via Smad-dependent and Smad-independent transcriptional pathways (Massague 2008). Thus Smad-mediated induction of Snail, Slug and Twist via HMGA2 (high motility group A2) and Smad-independent phosphorylation of Par6 contribute to dissolution of cell junction complexes (Ozdamar, Bose et al. 2005; Thuault, Valcourt et al. 2006). Furthermore, EMT recruits the cooperation between oncogenic Ras and receptor tyrosine kinases (RTKs) to induce downstream Raf/MAPK signaling associated with tumor progression and poor clinical diagnosis (Grunert, Jechlinger et al. 2003). The loss of cell polarity is a crucial step for EMT. Par, Crumbs, and Scribble protein complexes were showed to participate in establishing and maintaining apicobasal polarity, and are regulated by EMT inducers. SNAIL1 alters epithelial cell polarity by repressing the transcription of Crumbs3 and abolishing the localization of both Par and Crumbs complexes at the junctions, Zeb1 represses the transcription of cell polarity genes (Crumbs3, Pals1-associated tight junction proteins (PATJ), and the member of the Scribble complex Lethal giant larvae (Lgl2)). TGFβ also contributes to the loss of cell polarity during EMT through the canonical pathway by inducing Snail and Zeb genes expression and through a noncanonical pathway that involves the downregulation of Par3 expression and the Par6-mediated degradation of RhoA and local alteration of the actin cytoskeleton (Thiery, Acloque et al. 2009). The important players in EMT which are investigate in this study are listed in Table 4.1.

Since tumor epithelial cells gain the ability to migrate and invade by dedifferentiating through activation of biological pathways associated with EMT, in this chapter, we investigated the involvement of the androgen signaling axis in EMT and invasive phenotype of prostate cancer cells. Our findings demonstrate that androgens

induce changes characteristic of EMT and cytoskeleton re-organization, involved in the metastatic behavior of castration-resistant prostate cancer cells.

Results Effect of Androgens on EMT Pattern of Prostate Cancer Cells Exposure of PC3 prostate cancer cells to DHT results in reduced expression of the epithelial markers, E-cadherin and β-catenin, and induction of the mesenchymal marker, N-cadherin expression (Fig. 4.1A); these are changes characteristic of EMT. LNCaP cells did not exhibit the same sensitivity as PC3 cells to DHT induced EMT; a significant reduction in E-cadherin and β-catenin was detected only after exposure to high doses of DHT (10nM) (Fig. 4.1A). However, the presence of TGFβ receptor II (TGFβRII) sensitizes LNCaP prostate cancer cells to the androgenic effect on EMT (0.1nM DHT) (Fig. 4.1A). Since nuclear translocation of β-catenin has been established as a significant event in EMT (Eger, Stockinger et al. 2000; Mulholland, Cheng et al. 2002), we subsequently analyzed the cytosolic and nuclear fractions of three different cell lines, LNCaP, LNCaP TβRII and C4-2B; we found that DHT triggered a marked nuclear translocation of β-catenin only in LNCaP TβRII cells. Consistent with the E-cadherin expression pattern (Fig 4.1A), DHT (1nM) failed to trigger β-catenin nuclear translocation in either the LNCaP cells, or the C4-2 cells (Fig 4.1B). In order to determine the transcriptional modulation of E-cadherin by DHT, quantitative PCR analysis was performed and downregulation of E-cadherin mRNA levels was detected in both PC3 and LNCaP TβRII cells (Fig 1E).

In view of the widely acknowledged role of TGFβ as a potent EMT inducer, the effect of TGFβ on prostate cancer cell EMT was examined as a positive/reference control. Exposure to DHT alone or in combination with TGFβ led to comparable reduction in E-cadherin and β-catenin levels in LNCaP TβRII cells (Fig. 4.1C). Since EMT is driven by the transcriptional factor Snail, which is upregulated by TGFβ (Massague 2008; Thuault, Tan et al. 2008), we subsequently investigated the effect of DHT on Snail expression. As shown on Figure 4.1D, treatment of LNCaP TβRII cells with DHT alone or in combination with TGF-β, led to a significant increase in Snail

expression. Furthermore, a marked induction in Snail expression by DHT was detected at the mRNA level in both LNCaP TβRII and PC3 cells (Fig 4.1F).

Androgens Affect Cytoskeleton Reorganization in Prostate Cancer Cells The process of cytoskeleton reorganization which directly affects cell migration and metastatic ability is a characteristic phenomenon in EMT. One of the critical proteins that promotes actin polymerization and defines the direction of cell motility is cofilin. Cofilin is a ubiquitous actin-binding factor required for the reorganization of actin filaments by causing depolymerization at the end of filaments and preventing their reassembly (Ghosh, Song et al. 2004; Meyer, Kim et al. 2005). Talin is another actin- binding protein that links integrins to the actin cytoskeleton in focal adhesion complexes and plays a role in cell adhesion and cell motility (Calderwood, Yan et al. 2002; Tanentzapf and Brown 2006). In order to study changes in cytoskeleton organization responses to androgens, the expression of key cytoskeleton components was evaluated by Western blot analysis and immunofluorescence staining. DHT treatment of LNCaP TβRII cells led to upregulation of β-actin and its partner cofilin, as well as the major focal adhesion effector, talin (Fig. 4.2A). Expression of α-tubulin was also significantly downregulated (Fig. 4.2A). Flow cytometric analysis revealed a significant increase in actin, talin and cofilin fluorescence density in cells after DHT treatment, compared to CSS-control cells (Fig. 4.2B). Furthermore, DHT exposure led to changes in actin cytoskeleton reorganization: prostate cancer cells exhibit more cytopodia and microvilli and share similar features with TGFβ treated cells. In addition, a large number of cells acquire a more round morphology in response to DHT and TGFβ treatment (Fig. 4.2C). Exposure to DHT for 3 days enhanced the association of actin with both cofilin and talin (Fig. 4.2D). A similar association was detected after short-term exposure (10mins) to DHT. These observations implicate an association between the actin microfilaments with cell motility and migration in response to androgens, possibly facilitating interaction with the ECM. Androgens and TGFβ Promote Prostate Cancer Cell Migration and Invasion Exposure of LNCAP TβRII cells to either DHT or TGFβ (as single treatment) significantly enhanced cell migration. Interestingly, the DHT/TGFβ combination did not lead to a synergistic increase in prostate cancer cell migration ability after 3 days

treatment (Fig. 4.3A), consistent with our EMT observations (Fig. 4.1 C and D). We subsequently examined the effect of DHT on prostate cancer cell invasion using the Boyden Chamber invasion assay. As shown on Figure 4.3B, DHT enhances the invasion ability of LNCaP TβRII cells, but has no significant effect on the parental LNCaP cells, consistent with our observation that low androgen levels (1nM DHT) failed to induce EMT in LNCaP cells and implicating an intact TGFβ signaling is required for the manifestation of the androgenic effect.

High AR Content Suppresses Androgen-induced EMT Phenotype PC3 cells exhibited a strong sensitivity to the EMT effect by DHT (Fig 4.1A). In order to determine the role of AR in androgen-induced EMT, we initially evaluated AR expression in PC3 cells using Western blotting and flow cytometric analysis. In accordance with the recent reports that AR is expressed in PC3 cells at low level (Alimirah, Chen et al. 2006; Martinez, Jasavala et al. 2008), we also found that prolonged exposure of Western blots revealed detectable AR levels (Fig. 4.4B). This was confirmed by FACS that revealed a marked peak shift in AR immunfluoresence, compared to the isotype IgG staining control, and AR expression could be induced by the DHT treatment (Fig. 4.4D). Considering the evidence that membrane located, non-classical AR could be activated by androgens to elicit multiple downstream effects (Cinar, Mukhopadhyay et al. 2007), we pursued the significance of membrane-associated AR in signaling the EMT effect. Figure 4A reveals that in PC3 cells treatment with BSA-conjugated testosterone (unable to go through cell membrane), failed to induce the EMT phenotype. However exposure of both LNCAP TβRII and PC3 cells to BSA-conjugated testosterone induces critical downstream signaling events including MAPK and Src activation (Fig. 4.7), implicating that non-genomic AR signaling might be involved in dictating EMT.

Considering that elevation of AR in PC3 cells suppressed the EMT phenotype, we subsequently determined whether or not the EMT effect requires AR function (ligand- induced). The EMT phenotype was profiled in the presence of the AR antagonist Casodex (10μM) in PC3 cells. As shown in Figure 4.4C, DHT-induced downregulation of E-cadherin and β-catenin (epithelial markers), and upregulation of N-cadherin (mesenchymal marker) was abolished (Fig 4.4C). The tissue type specificity of androgen- induced EMT was investigated in AR-bearing human breast cancer cells MCF-7 and

renal cell carcinoma cells, 786-0. In response to DHT, the EMT phenotype was evident in MCF-7 breast cancer cells but not in renal cancer cells (Fig. 4.9).

The function of AR in mediating the EMT effect was determined by introducing the wild type (wt) AR and the mutant (mt) AR [(877A mutation) with higher androgen affinity], in PC3 cells (low endogenous AR) (Fig 4.5A). Overexpression of either the wtAR or mtAR (LNCaP harbored AR mutation), in PC3 cells significantly suppressed their growth (Fig. 4.8). The expression pattern of E-cadherin, β-catenin and N-cadherin was evaluated after exposure of cells to DHT (1nM). As shown on Figure 4.5C, DHT led to decreased expression of E-cadherin and β-catenin, and upregulation of N-cadherin, changes characteristic of EMT, in the parental PC3 cells, but not in AR overexpressing PC3 cells.

Subsequent experiments examined the effect of DHT on cell migration and invasion in PC3 AR overexpressing cells. DHT enhanced the invasion ability in PC3 parental cells, while there was no effect on either the wt or the mtAR overexpressing cells (Fig. 4.6A). To trace androgen-regulated changes in the cytoskeleton reorganization in prostate cancer cells, the intracellular localization and distribution of cofilin and β-actin were determined in response to DHT. As shown on Figure 4.6C, in response to DHT, parental PC3 cells exhibited marked changes in the actin cytoskeleton organization and cofilin/actin colocalization, resembling the EMT characteristics, while AR overexpressing PC3 cells failed to exhibit any such changes.

Low AR Content Sensitizes Prostate Cancer Cells to Androgen-induced EMT The AR requirement in androgen-induced EMT was examined by loss-of- expression studies. AR expression was effectively suppressed in LNCaP and CWR22 cells using the shRNA approach (Fig 4.5B). The expression pattern of E-cadherin and β- catenin was used to evaluate the EMT effect in both LNCaP and CWR22 cells. DHT (1nM) failed to induce EMT in the parental LNCaP or CWR22 cells (Figs. 4.1B, 4.5D). In cells harboring low AR content, DHT induced downregulation of E-cadherin and β- catenin (Fig. 4.5D). Immunofluorescence analysis revealed the actin cytoskeleton reorganization and the enhancement of the co-detection of actin filament and cofilin/talin in the AR-silenced cells, but not in parental control cells (Fig. 4.6D). In addition, DHT

increased the invasion potential of LNCaP AR-silenced cells, while there was no significant change in the LNCaP parental cell invasion in response to DHT (Fig. 4.6B). Thus low intracellular AR levels sensitize prostate cancer cells to androgen-induced EMT.

Discussion The functional outcome of EMT in prostate cancer progression to castration- resistant disease is likely to be complex, given the uncertainty surrounding the contribution of the androgen axis to prostate cancer metastasis. Indeed the impact of androgen suppression to metastatic dissemination of prostate cancer cells is still a subject of debate, with the notion that androgen deprivation therapy may downregulate AR in prostate tumors. One could speculate that a threshold low AR level may promote EMT, ultimately facilitating metastatic spread of prostate tumor epithelial cells. The inhibition of EMT response to androgens by AR overexpression, points to: (a) an inverse relationship between AR content and EMT induction and (b) a potential biochemical basis for the metastatic behavior of prostate cancer cells from recurrent castration- resistant tumors. Since long term androgen deprivation may downregulate AR expression, this threshold of “low” AR status facilitates DHT induced EMT, thus promoting cancer metastasis. This is in accord with our observations that the AR antagonist reverses the EMT changes triggered by androgens in prostate cancer cells thus providing proof-of-principle as to the ability of elevated AR to prevent DHT-induced E- cadherin reduction and N-cadherin-induction. According to our data, the wildtype and 877A mutation AR present the same manner in the regulation of EMT induction. This indicates that 877A site in AR is not important to androgen induced EMT. The concept gains indirect support from the clinical evidence that intermittent androgen deprivation therapy benefit patients in prostate cancer progression (Boccon-Gibod, Hammerer et al. 2007). Emerging data from an ongoing clinical trial shows intermittent androgen deprivation therapy to be a promising option for patients with locally advanced and metastatic prostate cancer, in accord with pre-clinical evidence suggesting that androgen

deprivation therapy (on the basis of intermittent administration) delays androgen independence (Gleave, Hsieh et al. 1993; Suzuki, Kamiya et al. 2008).

Pulse administration can effectively target AR regulation, providing proof-of- principle that low AR levels induced by androgen deprivation therapy might be responsible for the more aggressive behavior of recurring prostate tumors and supporting the requirement of a threshold AR level to maintain prostate tumor growth. Gain-of- function studies have shown that activated AR (via mutational activation or ligand independent activation) promotes proliferation of prostate cancer cells (Burnstein 2005; Balk and Knudsen 2008). In a “double-sword” twist, the present data suggest that loss of AR can actually promote prostate cancer cell metastatic ability by regulating EMT. This study provides a novel insight into the androgen-mediated EMT effect, as a biological process significantly contributing to castration-resistant prostate cancer metastasis.

Figure 4. 1 Effect of Androgens on EMT of Prostate Cancer Epithelial Cells.

A, B and C) Prostate cancer cells (PC3, LNCaP and LNCaP TβRII) were treated with DHT (0.1-10nM) as shown for 72 hrs. Total cell lysates were analyzed by Western blotting to determine the expression of E-cadherin, β-catenin and N-cadherin. D) LNCaP, LNCaP TβRII and C4-2B cells were treated with DHT for 72hrs and subjected to subcellular fractionation as described in “Materials and Methods”. Western blot analysis was performed in cytosolic and nuclear fractions to determine β-catenin levels. GAPDH and PARP served as an internal control for cytosolic and nuclear fractions respectively. E and F) LNCaP TβRII and PC3 cells were treated with DHT (0.1-10nM) for 24hrs and relative mRNA expression level of E-cadherin and Snail was evaluated using real-time PCR. The mean value is determined by triplicate wells. *, P<0.05

Figure 4.1

Figure 4. 2 Androgens Regulate Cytoskeleton Reorganization of Prostate Cancer Cells.

A) LNCaP TβRII cells were exposed to DHT (1nM) and/or TGFβ (5ng/ml). Expression of actin, talin ,cofilin and tubulin was determined by Western blot analyais. GAPDH served as internal loading control. B) LNCaP TβRII cells were exposed to DHT and subjected to immunofluorescence staining for actin, talin and cofilin detection. The level of cytoskeleton proteins was assessed by FACS. C) LNCaP TβRII cells were treated with DHT and/or TGFβ and F-actin was detected using FITC-phalloidin under fluorescent microscopy. Arrows indicate microvilli formation. D) Following treatment with DHT, LNCaP TβRII cells were subjected to immunofluorescence: red color indicated cofilin and talin (red) respectively in E and F; green color indicated actin, blue color shows nuclear staining, yellow color indicates co-detection.

Figure 4.2

Figure 4. 3 Effect of Androgens and TGFβ on Prostate Cancer Cell Invasive Behavior.

A) LNCaP TβRII cells were treated with DHT (1nM) and/or TGFβ (5ng/ml) for 24, 48 and 72hrs and cell migration was determined. B) LNCaP and LNCaP TβRII cells were exposed to DHT for 48hrs and cell invasion was assessed as described in “Materials and Methods”. Numerical values indicate the average of three experiments +/- SEM ( standard error of the mean). *, P<0.05.

Figure 4.3

Figure 4. 4 Detection of AR in PC3 Cells.

A) PC3 cells were treated with increasing doses (0.1, 1 and 10nM) of BSA-testosterone. Expression pattern of β-catenin, E-cadherin and N-cadherin were evaluated by Western blotting. GAPDH was used as a loading control. B) AR expression in PC3 cells after DHT-treatment was detected by Western blot. C) PC3 cells were treated with DHT (0.1- 10nM) and Casodex (10μM). Expression of E-cadherin, β-catenin, N-cadherin and vimentin was addressed by western blot. D) AR expression in PC3 cells after DHT- treatment determined by immunofluorescence staining followed by FACS analysis.

Figure 4.4

Figure 4. 5 AR Status and EMT.

A) Wildtype AR and LNCaP Mutant AR are stably transfected onto PC3 cells. The expression of AR is detected by western blotting in these cell lines. B) AR ShRNA was transfected into LNCaP cells and CWR22 cells and stable transfectants were generated. The loss of AR protein expression was examined by Western blotting. C) PC3 Zeo, PC3- hAR and PC3-LAR cells were treated with DHT (1nM) and/or TGFβ (5ng/ml). Expression of E-cadherin, β-catenin, and N-cadherin were determined by western blotting. GAPDH served as internal control. D) LNCaP null vector control cells and LNCaP AR sh cells; CWR22 null vector control cells and CWR22 AR sh cells were treated by DHT (1nM) and TGFβ (5ng/ml) and expression of E-cadherin and β-catenin was assessed by Western blotting.

Figure 4.5

Figure 4. 6 AR Involvement in EMT-related Cytoskeleton Reorganization and Cell Invasion.

A) PC3 Zeo, PC3-hAR and PC3-LAR cells were treated with DHT and their invasion ability was assessed. B) Effect of AR loss on the invasion ability of prostate cancer cells. LNCaP null vector control cells and LNCaP AR sh cells; CWR22 null vector control cells and CWR22 ARSh-silenced cells were exposed to androgens and their invasion potential was determined. C) PC3 Zeo, PC3-hAR and PC3-LAR Cells were treated with DHT and subjected to immunofluorescence analysis as described in “Materials and Methods”; cofilin (red), green indicates actin microfilaments; nuclei detected by blue staining. D) LNCaP null vector control cells and LNCaP AR sh cells; CWR22 null vector control cells and CWR22 AR sh cells were treated with DHT and immunofluorescence analysis for actin (green), cofilin and talin (red) was conducted. Numerical values indicate the average of three experiments +/- SEM ( standard error of the mean). *, P<0.05.

Figure 4.6

Figure 4. 7 Effect of Non Genomic-BSA-conjugated Testosterone on EMT Signaling in Prostate Cancer Cells.

PC3 cells and LNCaP TβRII cells were treated with 0.1nM, 1nM and 10nM BSA- testosterone conjugate. Protein expression levels of key signaling effectors, cdc1, MAPK, Src and p-Src were evaluated by western blotting and were normalized to GADPH expression.

Figure 4.7

Figure 4. 8 Overexpression of AR inhibits PC3 cell viability.

The growth rate of PC3 Zeo null vector control cell line and AR overexpressing cell lines was determined using the MTT assay. Overexpression of both the wild type AR and the AR harboring the mutation found in LNCaP cells led to a significant loss of cell viability in prostate cancer cell PC3 compared to controls. Numerical values indicate the average of three experiments +/- SEM ( standard error of the mean). *, P<0.05.

Figure 4.8

Figure 4. 9 Androgens Induce EMT in Human Breast Cancer Cells but Not in Renal Carcinoma Cells.

Human breast cancer cells, MCF-7 and human renal cancer cells, 786-0 cells were treated with increasing doses of DHT (0.1, 1.0 and 10nM DHT), E expression levels of E- cadherin, β-catenin and N-cadherin were evaluated by Western blotting; GAPDH is used as internal loading control.

Figure 4.9

Table 4. 1 Molecules Involved in EMT

CHAPTER FIVE CHEMOTHERAPY-BASED MICROTUBULE TARGETING SUPPRESSES ANDROGEN RECEPTOR ACTIVITY IN PROSTATE CANCER CELLS

Introduction Considerable efforts have been invested towards a better understand the targets and molecular mechanisms contributing to prostate cancer progression. PSA level is a specific marker for androgen axial signaling which is highly correlated with prostate cancer progression: the lower of PSA indicated the good prognosis for prostate cancer. Rising prostate specific antigen (PSA) levels can serve as an indication that AR activity is inappropriately restored in castration-resistant cancer (CRPC).

AR is composed of several major domains: N-terminal transcription-activation function-1 (AF-1) region, the central DNA-binding domain (DBD) and the C-terminal ligand-binding domain (LBD) and a hinge region. Within the nucleus, AR scans the genome for androgen-response elements (AREs) in the promoters and enhancers of target genes, and recruits factors that are necessary for transcription. There is numerous evidence indicates that androgens regulate the location of ARs in the cell. ARs have steady-state nuclear distribution in prostate cancer xenograft cells unless the host is castrated, in which case ARs adopt a predominantly cytoplasmic distribution (Zhang, Johnson et al. 2003). In many cells in tissue culture (androgen-free culture medium), ARs are predominantly cytoplasmic and undergo nuclear import in response to androgen. Cytoplasmic forms of nuclear receptors exist in complexes with chaperones, including heat-shock protein 90 (Hsp90) and Hsp70 (Jenster, Trapman et al. 1993; Cutress, Whitaker et al. 2008). Nuclear import and nuclear export of AR involve more than one export signal. It was reported that DBD of the AR is sufficient to specify nuclear export and point mutations in the DBD inhibit nuclear export of full-length AR (Black, Holaska et al. 2001; Black, Vitto et al. 2004). recent evidence also shows that there is a nuclear-export signal in the LBD of the AR(Saporita, Zhang et al. 2003). There is also evidence that the N-terminal AF-1 region contributes to subnuclear targeting of the AR. ARs are highly mobile in the nucleus, agonist-bound ARs are concentrated transiently in

a subnuclear compartment that has the appearance of fine granules (Black and Paschal 2004).

The tubulin/microtubule system is an integral component of the cytoskeleton. Microtubules are highly dynamic structures that play a critical role in orchestrating the separation and segregation of chromosomes during mitosis. This makes microtubules highly valued as anticancer drug targets. Tubulin-binding agents are derived from natural sources and include a large number of agents with diverse chemical structures. What all tubulin-binding agents share in common is their ability to disrupt microtubule dynamics, induce mitotic arrest and cell death. The best known of these agents are the vinca alkaloids and taxanes, which at high doses cause microtubule destabilisation and microtubule stabilisation, respectively. Two independent Multicenter phase III studies (Southwest Oncology Group (SWOG) 99-16 and TAX 327) compared Taxane-based regimens with mitoxantrone/prednisone and demonstrated a significant survival benefit in patients. (Petrylak, Tangen et al. 2004; Tannock, de Wit et al. 2004). Docetaxel, a semi synthetis taxane, stabilized the microtubule through binding on β-actin. Once bound with taxanes, microtubules cannot be disassembled and this static polymerization disrupts the normal mitotic process, arrests cell in the G2M cycle phase and ultimately leading to apoptosis. (Kraus, Samuel et al. 2003). Docetaxel and prednisone chemotherapy had become first-line standard therapeutic regimens of metastatic androgen independent prostate cancer treatment. The evaluation of use this therapy in early stages of prostate cancer and in combination with other chemotherapy regimen is still under investigation (Mancuso, Oudard et al. 2007).

Androgen deprivation is the only clinically effective therapy for the treatment of advanced metastatic prostate cancer currently. After the initial response, however, there is tumor recurrence in the majority of patients, due to emergence of androgen-independent state (Shahinian, Kuo et al. 2005). Androgen-induced prostate epithelial cell proliferation is regulated by an indirect pathway involving paracrine mediators produced by stromal cells, such as insulin-like growth factor (IGF), fibroblast growth factor (FGF) and epidermal growth factor (EGF) (Cunha and Donjacour 1989; Byrne, Leung et al. 1996). The absence of a link between elevated serum testosterone, DHT, or adrenal androgens and prostate cancer risk, suggests that androgens are not sufficient to promote prostate

carcinogenesis (Roberts and Essenhigh 1986; Hsing 2001). Prostate tumors however eventually recur due to a transition from androgen-dependent tumor growth to a highly metastatic and androgen-independent disease for which there is no effective therapy available. The long-term benefit of androgen deprivation in patients with metastatic disease has been the subject of debate (Makhsida, Shah et al. 2005; Shahinian, Kuo et al. 2005; Lu-Yao, Albertsen et al. 2008). Recent breakthroughs in the development of novel AR-antagonist strategies have lead to Phase 1 clinical trials with the potential to improve the efficacy of AR targeting and therapeutic outcome in patients with CRPC (Tran, Ouk et al. 2009).

Paralleling these reports is the realization that the non-mitosis related function of taxane can target prostate cancer. Docetaxel counteracts the pro-survival effects of Bcl-2 gene expression (Debes and Tindall 2004; Oliver, Miranda et al. 2005). Bcl-2 gene is part of class of oncogenes that contributes to neoplastic progression by inhibition of apoptotic cell death and the phosphorylation of Bcl-2 protein leads to loss of Bcl-2’s antiapoptotic function (Kraus, Samuel et al. 2003). However, the effect of taxanes on the AR signaling axis during prostate cancer progression and emergence of therapy-resistant prostate tumors, remains unknown. This chapter provide the first study of the involvement of tubulin/microtubule organization in AR signaling during prostate cancer progression.

Results Taxol Chemotherapy Inhibits PSA Expression in Prostate Cancer Taxol chemotherapy reduces the serum PSA levels in prostate cancer patients (Beer, Pierce et al. 2001; Berry, Dakhil et al. 2001). To investigate whether the reduction in PSA is due to either tumor shrinking or impairing the signaling axis, PSA expression was profiled in prostate cancer epithelial cells, by performing immunocytochemical analysis using the TMAs of human prostate specimens from docetaxel-treated prostate cancer patients. The results shown on Figure 5.1 reveal the ability of docetaxel to inhibit PSA expression in individual prostate tumor cells (Fig. 5.1A). Quantitative analysis of the data shows a significant reduction (19%) in the intensity of PSA in prostate tumors

from patients receiving docetaxel, compared to specimens from untreated patients (Fig. 5.1 B).

Taxol Inhibits AR Transcriptional Activity Paclitaxel and Nocodazole were used to disrupt normal cellular function of microtubule system. Similar with docetaxel, Paclitaxel is chemotherapy drug classified with the taxane group and used in the treatment of advanced prostate cancer and recurrent prostate cancer (Mancuso, Oudard et al. 2007). Nocodazole exerts its effect in cells by interfering with the polymerization of microtubules. Subsequent experiments focused on determining the effect of microtubule targeting drugs on AR activation in vitro. The mRNA levels of PSA were evaluated by quantitative PCR in response to DHT/ microtubule targeting drugs. Treatment of human prostate cancer LNCaP cells with DHT (1nM) for 24hrs led to a significant increase in the expression of PSA mRNA. Nocodazole completely abolished and paxlitaxel partially inhibited this PSA induction (Fig. 5.2 A). The changes in PSA protein levels were consistent with the mRNA changes in response to treatment (Fig.5.2B). To further investigate the consequences of microtubule targeting on AR transcriptional activity, the ARE-luciferase vector was introduced to LNCaP cells in response to DHT in the presence of Nocodazole or Paclitaxel. Activation of ARE was detected within 24hrs of DHT treatment and was significantly inhibited by both drugs (Fig. 5.2 C).

Taxol Inhibits Ligand-independent AR Transcriptional Activity It was previously reported that EGF induces ligand-independent AR activation in prostate cancer cells with hypophysical androgen level (Oosterhoff, Grootegoed et al. 2005). To determine the effect of microtubule targeting drugs on ligand-independent transcriptional activation of AR, EGF was used to induce the androgen- independent activation of AR. A significant increase in PSA mRNA expression was detected in response to EGF in combination with DHT, while Nocodazole or Paclitaxel ablated this PSA mRNA induction within 24hrs (Fig. 5.3 A). To investigate whether the impaired AR transcriptional activity is specific to microtubule targeting drugs, two different drugs, Velcade and Doxazosin were examined (Fig.5.3 B). Exposure to either one of these agents did not affect the androgen-mediated PSA mRNA expression (Fig.5. 3 C).

Microtubule Targeting Chemotherapy Inhibits AR Nuclear Translocation In order to further investigate the effect of taxol drugs on AR function in prostate cancer cells, the expression levels of AR was evaluated in docetaxel-treated prostate cancer patients. There was no significant change of AR expression level in prostate epithelial cells between the two groups (Fig. 5.4A). However marked changes in the cellular localization of AR were observed after taxol treatment. For the prostate specimens derived from patients untreated, 50% prostate cancer epithelial cells exhibited nuclear accumulation of AR, while only 10% of the cell population had cytoplasmic localization of AR (Fig. 5.4B and C). For Docetaxel-treated patients, there was a marked reduction in nuclear translocation of AR (to 38%), with a parallel increase (to 29%) predominantly in the cytoplasm (Fig. 5.4B and C). The AR localization also correlated with PSA expression level in prostate epithelial cells. Cells with nuclear AR localization exhibited a higher PSA expression (Fig.5.4D). To determine the impact of microtubule targeting on AR localization in vitro, immunofluorescence staining was conducted to evaluate the AR nuclear translocation in response to taxol treatment in prostate cancer cells. As shown on Figure 5.5A and B, DHT treatment (4hrs) induces a robust AR nuclear translocation in LNCaP cells. Pre-treatment of Paclitaxel and Nocodazole for 24hrs abrogated this AR nuclear translocation as shown by fluorescent microscopy (Fig. 5.5A and B). Western blot analysis of the cellular compartments after subcellular fractionation also revealed that DHT- induced nuclear translocation of AR was blocked after Paclitaxel and Nocodazole treatment (Fig. 5.5C).

The process of EMT during which cells lose their polarity and cell-junction proteins and acquire mesenchymal cell markers is linked to tumor progression and metastasis (Thiery et al, Cell, 2009). Since our earlier studies demonstrated that androgens via the AR regulate EMT and cytoskeleton organization involved in the invasive behavior of prostate tumor epithelial cells, we next examined the consequences of taxol-chemotherapy on EMT. Expression of E-cadherin, β-catenin (epithelial cell markers) and N-cadherin (mesenchymal cell marker), was immunohistochemically profiled in the prostate TMAs from treated vs. untreated patients. The data reveal that Doxetaxel treatment had no significant impact on EMT (Figure 5.8).

Tubulin Interacts with the AR

Microtubule is the main cytoskeleton protein component responsible for intracellular protein transportation, and facilitates many cellular events. The potential interaction between AR and microtubules was subsequently investigated. Interaction of endogenous AR and α-tubulin was detected in both LNCaP and CWR22 cells (Fig.5.5D and E). The co-detection of AR and tubulin was detected by immunofluoresce staining as yellow color dramatically reduced by DHT treatment (Fig. 5.5D and E; Figure 5.7). To further determine the interaction site of AR with tubulin, different truncated forms of AR (Fig. 5.5F) were transfected in prostate cancer PC3 cells, which harbor very low endogenous AR. Loss of C-terminal domain and hinge domain cannot inhibit the interaction of AR and tubulin, so this indicated that the N-terminal domain is responsible for the AR and tubulin association and potential interaction (Fig. 5.5G).

Androgens Downregulate Tubulin in Prostate Cancer Cells To determine whether androgen signaling can impact the microtubules, tubulin expression was evaluated by Western blot analysis and immunofluorescence. Treatment of prostate cancer cells with DHT significantly inhibited tubulin expression (Fig. 5.6A). There was a marked reduction in tubulin levels, an effect that was enhanced by TGFβ (Figure 5.7). Immunofluorescence staining revealed that the microtubule spindles were undetectable after androgen treatment (Fig. 5.6B).

Discussion Microtubules are polar cytoskeletal filaments assembled from head to tail and lateral associations of a/b-tubulin heterodimers. The motor protein Kinesin-1 is recruited to the microtubule and preferentially moves various cargoes, including vimentin filaments and transferin, along detryosinated microtubules (Liao and Gundersen 1998; Kreitzer, Liao et al. 1999). Furthermore, the microtubule network has been implicated in facilitating the nuclear import of several cancer regulator proteins, such as pTHrP, P53 and Rb (Giannakakou, Sackett et al. 2000; Jiao, Datta et al. 2006; Roth, Moseley et al. 2007).

Ample evidence suggests that both androgen dependent and independent activation of AR required the nuclear translocation to perform the downstream function

of androgen signaling (Feldman and Feldman 2001). Not only the expression level of AR but also the cytoplasmic “zip code” are critical for prostate cancer progression. The interruption of AR nuclear localization and transcriptional activity by microtubule stabilizing chemotherapy drug is demonstrated in this study. Considering the interaction of AR and tubulin, our data raise the possibility that preferential binding of microtubules to AR could lead to the recruit of active forms AR to facilitate its transcriptional activity. Since the nuclear protein import is not generally dependent on microtubules (Roth, Moseley et al. 2007), and the AR inhibition was not observed in other chemotherapy drugs than microtubule targeting drugs, the effect of AR inhibition by microtubule targeting drug might be very specific. This illustrated a novel function for microtubule target chemotherapy drugs in regulating the subcellular localization of AR in prostate cancer cells.

Modification of tubulin (detryosination /tyrosintion) affect the microtubule stability (Hammond, Cai et al. 2008). Our study shows the binding of α-tubulin and AR which is necessarily through N-teminal domain of AR. It was reported that estrogens regulate β-tubulin synthesis and decrease the density of microtubules and block cells at G2M in prostate cancer (Bonham, Galkin et al. 2002; Montgomery, Bonham et al. 2005). The binding of activated AR is a potential modifier of microtubule. The evidences that androgen suppresses the expression of α-tubulin and downregulate the microtubule in prostate cancer cells point to a negative feedback regulation in microtubule- AR interaction. Androgen signaling is an important cell differentiation factor and regulates cell cycle including G2M arrest (Fig 5.10) which is consistent on function with its inhibition of microtubule. The suppression of tubulin gene transcripts could potentially be due to several different levels of regulation. Typically, when the proportion of soluble tubulin heterodimers increases in the cell, gene transcription is modulated in order to maintain homeostatic level of free and polymerized tubulin (Cleveland, Lopata et al. 1981). The directly transcriptional regulation could also contribute to this inhibition. The quantitately evaluation of the amount of polymerized and unpolymerized tubulin in these cells with androgen and/or Paclitaxel could be a nice approached to explain the underling regulation mechanism. The evidence that microtubule targeting drugs inhibits the

androgen/AR axis signaling points to a potentially enhanced therapeutic value for combination of anti-androgen therapy with taxanes in the treatment of prostate cancer.

Figure 5. 1 Doclitaxel Suppresses PSA Expression in Human Prostate Tumors

A) PSA expression profile in prostate tumor epithelial cells in specimens from doclitaxel- treated and untreated prostate cancer patients. B) Quantitative analysis of PSA immunoreactivity pattern in prostate tissue arrays: from the left panel, untreated patients; right, Doclitaxel-treated patients. Mean value of all the samples are calculated, error bar indicated the standard error. *P<0.05

Figure 5.1

Figure 5. 2 Microtubule Targeting Drugs Inhibit Ligand-dependent AR Transcriptional Activity

LNCaP cells were treated with DHT (1nM) in the presence or absence of Nocodazole (5ug/ml) or Paclitaxel (1μM). A) PSA mRNA expression was determined by realtime PCR. B) PSA protein levels were assessed by Western blotting and relative expression was quantitated (lower panel). C) The ARE luciferase reporter vector was introduced into LNCaP cells and AR transcriptional activity was determined using the luciferase assay. Mean value of all the samples are calculated, error bar indicated the standard error. *P<0.05

Figure 5.2

Figure 5. 3 Microtubule Targeting Inhibits Ligand-independent AR Transciptional Activity

A) LNCaP cells were treated with a combination of DHT (0.1nM) and EGF (5nM) with or without Nocodazole (5μg/ml) or Paclitaxel (1μM). AR transcriptional activity was evaluated with PSA realtime PCR. B) LNCaP cells were treated with the following chemotherapeutic agents for 24-72hrs: TRAIL, Velcade, Doxasosin, Nocodazole (5μg/ml) or Paclitaxel (1μM) and cell death was determined using the MTT assay. C) LNCaP cells were treated with DHT (1nM), in the presence or absence of Velcade or Doxasosin as shown. PSA mRNA expression was evaluated using Real-time PCR. * P<0.05

Figure 5.3

Figure 5. 4 Doclitaxel Suppresses AR Nuclear Translocation

A) AR protein expression levels in prostate cancer epithelial cells of Doclitaxel treated and untreated patients was evaluated by immunohistochemical staining. Three different areas were randomly selected and AR immnoreactivity was assessed as described in the method section. B) reveals a representative image of the subcellular AR localization in human prostate tissue: left panel, tissue from untreated patients; right, tissue from doclitaxel-treated patients. C) indicates the percentage of nuclear and cytoplasmic AR in Doclitaxel-treated and untreated tumors. D) reveals that AR localization correlated with PSA levels in prostate epithelial cells. Mean value of all the samples are calculated, error bar indicated the standard error. *P<0.05

Figure 5.4

Figure 5. 5 Tubulin Interacts with AR

A) and B) Androgens induce AR nuclear translocation in LNCaP cells and pre-treatment of Paclitaxel and Nocodazole for 24hrs abrogated this AR nuclear translocation. Subcellular localization of AR was detected by fluoresencent staining (red) (40x magnification). C) Western blot analysis of the cellular compartments after subcellular fractionation also revealed that DHT-induced nuclear translocation of AR was blocked in response to Paclitaxel or Nocodazole treatment. GAPDH and PARP were used as loading controls. D) and E) LNCaP TβRII and CWR22 cells, respectively were treated with DHT (1nM), in the presence or absence of TGFβ (5ng/ml). Immunoprecipitation was performed by using the antibodies against either tubulin or AR to show the AR- tubulin association). F) Truncated forms of AR transfected in PC3 cells. G) Immunoprecipitation analysis of the AR and tubulin interaction indicates that loss of ligand-binding and DNA-binding domain and hinge domain did not inhibit the AR- tubulin association.

Figure 5.5

Figure 5. 6 Androgens Inhibit Tubulin Expression in Prostate Cancer Cells

A) LNCaP, LNCaP TβRII cells and CWR22 cells were treated with DHT (1nM) for 72hrs. Tubulin levels were evaluated by Western blot. GAPDH was used as internal control. B) LNCaP TβRII cells and CWR22 cells were treated with DHT (1nM) with or without TGFβ (5ng/ml) for 72hrs. Tubulin expression was detected by immunofluoresnce (red); nuclei were stained by DAPI (blue).

Figure 5.6

Figure 5. 7 Suppression of Tubulin Expression by DHT and/or TGF β Treatment

LNCaP cells were treated with DHT (0.1-10μM) or in combination with TGF-β1 (5ng/ml) for 24-72 hrs. Tubulin expression was evaluated with Western blot. GAPDH was used as internal control.

Figure 5.7

Figure 5. 8 Expression Pattern of EMT Markers

The expression of E-cadherin (A), β catenin (B) and N-cadherin (C) are based on immunoreactivity in prostate TMAs. Numerical values indicate the relative immuneactivity for each specific marker protein from three different areas. D) LNCaP cells were treated with DHT alone (1ng/ml), or in combination with either Paclitaxel or Nocodazole for 24 or 48hrs, and expression of the EMT markers (E-cadherin, β catenin and N-cadherin), as well as AR was evaluated by Western blotting using the specific antibodies. GAPDH was used as internal control. E) Quantitative analysis shows the relative expression level of each protein after normalizing with GAPDH.

Figure 5.8

Figure 5. 9 Expression Pattern of Cell Cycle Markers

LNCaP cells were treated with DHT (0.1-10 nM) or in combination with TGFβ (5ng/ml) for 72hrs and the cell cycle markers cdc2, cdc25c, cyclin E, CDK2 were determined by Western blot analysis, with GAPDH serving as the internal control.

Figure 5.9

Figure 5. 10 Microtubule Facilitate AR Nuclear Translocation in Prostate Cancer Cells

On the basis of our finding so far, we propose the following mechanistic scenario: microtubules facilitate AR nuclear translocation and enhance downstream AR transcriptional activity in prostate cancer cells. Microtubule targeting chemotherapy blocks this pathway and suppresses AR signaling, via a negative feedback mechanism; AR signaling can inhibit tubulin expression thus impairing the cytoskeleton structure and organization.

Figure 5.10

CHAPTER SIX

DISCUSSION

Androgen Axis Cross-Talk with TGFβ Signaling in Prostate Tumorigenesis Emergence of hormone refractory prostate cancer has been attributed to AR mutations or amplifications that lead to a resistance to anti-androgens. AR mutations unable to activate androgen-responsive genes or change the sensitivity of the AR to circulating androgens may suppress androgen dependence (Zhao, Malloy et al. 2000), thus contributing to prostate tumorigenesis. Indeed, increased AR levels can confer resistance to antiandrogens by amplifying signal output from low levels of residual ligand (Chen, Welsbie et al. 2004). Another possible scenario involves prostate tumors that retain intact AR signaling but harboring changes in the AR co-regulators (coactivators/co-repressors) that cause ligand-independent AR activation. Moreover, androgen-resistance prostate tumor development can be driven by apoptosis/survival regulatory functions that bypass AR, such as growth factor signaling pathways, rendering the AR irrelevant to the development of androgen-independent cancer. Responsibility for such AR bypassing towards androgen-independent disease has been assigned to bcl-2 overexpression (McDonnell, Troncoso et al. 1992) and AR methylation status (Jarrard, Kinoshita et al. 1998; Kinoshita, Shi et al. 2000).

Androgens via the AR, play a critical mechanistic role in the deregulation of TGF-β signaling in prostate tumorigenesis, and TGFβ intracellular signaling effectors, (Smads 3, 4) serve as negative regulators of AR-mediated transcription in prostate cancer cells (Hayes, Zarnegar et al. 2001; Kang, Lin et al. 2001; Chipuk, Cornelius et al. 2002). Disruption of TGF-β1 signaling can contribute to androgen-independence and metastasis via cross-targeting AR, bcl-2 and β-catenin (Bruckheimer and Kyprianou 2002; Chesire, Ewing et al. 2002; Chipuk, Cornelius et al. 2002; Tu, Thomas et al. 2003; Ayala, Dai et al. 2006). Our previous studies demonstrated that androgens enhance the apoptotic effects elicited by TGF-β in prostate tumor epithelial cells LNCaP-TβRII harboring a mutant AR (Bruckheimer and Kyprianou 2001). This synergistic effect by androgens on TGFβ

mediated apoptosis is influenced by the status of AR activity. It was reporter earlier that in PC3 prostate cancer cells engineered to overexpress the wild type AR, the apoptotic action of TGFβ is not enhanced by androgens (Zhu, Fukada et al. 2006). Consistent with a specific activity of the LNCAP mutant AR as an apoptosis promoter, is recent evidence that AR has the ability to sensitize LNCaP prostate cancer cells to taxane-induced apoptosis (Hess-Wilson, Daly et al. 2006). Significantly, prohibitin (“new” TGFβ mediator) has been shown to inhibit prostate tumor growth by repressing AR activity (Gamble, Chotai et al. 2007).

Androgens can enhance TGF-β1 induced apoptosis of the androgen sensitive and TGF-β1 responsive prostate cancer cells LNCaP (containing mtAR)(Bruckheimer and Kyprianou 2001). This synergistic effect is blocked by bcl-2 overexpression (Bruckheimer and Kyprianou 2002). The present study provides new evidence that mutant AR nuclear translocation is induced in response to TGFβ alone, suggesting that TGFβ regulates AR transcriptional activity in a ligand independent pathway. The results suggest that this apoptotic promotion by androgens is dependent on AR status with the mutant AR selectively driving the apoptosis promoting effect of androgens while a wt AR can function as a suppressor of TGF-β-induced apoptosis in prostate cancer cells. TGFβ regulates androgen responsive genes in synergy with androgens, the cellular response might be different, depending on the AR status; mtAR dictates an apoptosis enhancing effect by androgens (Bruckheimer and Kyprianou 2001), while wtAR antagonizes the apoptotic effects of TGFβ in prostate cancer cells. Restoration of a TGFβ signaling pathway in prostate cancer cells suppresses prostate tumorigenic growth in vitro and in vivo by inducing apoptosis (Guo and Kyprianou 1999). Constitutively activated AR may cause loss of androgen dependence (i.e. apoptosis rescue and survival advantage) partially via loss of TGFβ signaling via inactivation of Smad 3 (Chipuk, Cornelius et al. 2002). The protein-protein interaction between AR and Smad4 may represent a new mechanistic link in the cross-talk between the TGFβ and AR signaling pathways, ultimately determining apoptosis outcomes. Considering the multiple binding partners for Smads and AR, the present findings provide a link between the androgen axis and TGFβ signaling, that may contribute to the emergence of androgen-independence during prostate tumorigenesis.

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Androgen Receptor Signaling and Tumor Microenvironment The precise role of the androgen axis and the impact of androgen-deprivation therapy in prostate cancer metastasis are still unclear. EMT is a process during which polarized epithelial cells acquire a migratory fibroblastoid phenotype and a critical event during cancer metastasis (Fuchs, Lichtenegger et al. 2002; Thiery 2002). The hallmark of EMT is loss of expression of the cell adhesion molecule E-cadherin. E-cadherin is cell- cell adhesion molecule that participates in calcium-dependent interactions to form epithelial adherent junctions. Prostate epithelial cells undergo EMT in response to an array of soluble factors including, TGFβ1 plus EGF, IGF-1, β2-microglobulin (β2-m), or exposure to a bone microenvironment (Zhau, Odero-Marah et al. 2008). The present findings demonstrate that androgens suppress E-cadherin and induce mesenchymal marker expression in prostate cancer epithelial cells. One could argue that this might facilitate escape of prostate cancer cells from the primary site and migration to distant sites, an important concept considering that activation of EMT may result in increased bone turnover, implicated in prostate cancer bone colonization in metastastatic disease.

Alterations in cytoskeleton reorganization induced by androgens may enable cell migration and metastasis of the escaped prostate tumor cells. Changes in actin microfilament network organization in androgen-treated cells could provide active movement assisting cell migration and the dynamics of interaction with adherent molecules in the ECM. Considering that the reactive prostate stroma has been assigned a critical role in the context of the tumor microenvironment in prostate cancer progression to metastasis, AR signaling in prostate fibroblasts may function as a promoter of prostate epithelial cell proliferation (Niu, Altuwaijri et al. 2008), as well as a mediator of a functional exchange between prostate epithelial and stromal cells, thus contributing to the EMT effect during cancer metastasis (Zhu and Kyprianou 2008).

The existence of crosstalk between androgen and TGFβ signaling has been established (Zhu and Kyprianou 2008). Interaction of Smad4, alone or together with Smad3, with the AR in the DNA-binding and ligand-binding domains, may result in the modulation of DHT induced AR transactivation. It was previously reported that in human

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prostate cancer cells PC3 and LNCaP, Smad3 enhances AR transactivation, while co- transfection of Smad3 and Smad4 repress AR transactivation (Bruckheimer and Kyprianou 2001; Kang, Lin et al. 2001; Kang, Huang et al. 2002). The interaction between the androgen axis and TGFβ signaling could be the determining factor for EMT manifestation. Nuclear translocation of β-catenin has been reported in the invasive front of colorectal carcinoma (Brabletz, Jung et al. 2001). Moreover, β-catenin activates DNA binding protein LEF-1/TCFs to induce several signaling pathways towards mesenchymal marker expression (Eger, Stockinger et al. 2000). A functional exchange between AR and β-catenin, results in increased nuclear colocalization and interaction of AR with β-catenin in castrate-resistant prostate tumors (Cronauer, Schulz et al. 2005; Wang, Wang et al. 2008). The present study suggests that activation of β-catenin by androgen signaling could serve as an alternative mechanism of androgen-induced EMT in prostate tumor epithelial cells. The involvement of several transcriptional factors (e.g. zinc-finger factors Snail and Slug, two handed zinc-finger factors ZEB1 and SIP1, and basic helix-loop-helix factors Twist and E12/E47) in the EMT process by repressing E-cadherin expression and consequently inducing migration and metastasis, has recently been documented (Peinado, Olmeda et al. 2007; Horiguchi, Shirakihara et al. 2009). Downstream activation of Snail by TGF-β/Smad pathway, represses E-cadherin expression in several cancer cell types (Nelson and Nusse 2004; Thuault, Tan et al. 2008). Androgen alone or in combination with TGFβ leads to a significant increase in Snail expression at both the mRNA and protein level in the androgen-sensitive, TGF-β-responsive LNCaP TβRII cells, suggesting that androgens can independently induce EMT, potentially bypassing the effect elicited by TGF-β. Thus DHT induces Snail expression in prostate cancer cells by engaging a cross-talk between the androgen axis and TGFβ signaling. Ongoing studies focus on the recruitment of β-catenin by Snail in EMT under conditions of androgen deprivation.

The microenvironment of the tumor cells which integrates the effect of growth factors, cytokines, hormones, extrocellular matrix and immune cells infiltration is the determineing factor for cancer growth and metastasis, as well as EMT. First, the host microenvironment includes soluble and insoluble factors associated with or secreted by osteoblasts, osteoclasts, marrow stromal, or stem cells that could play key roles promoting EMT, an important molecular transition by which cancer cells gain increased

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metastatic potential in response to the changing tumor microenvironment(Sung, Hsieh et al. 2007). These interactions could result in the promotion of cancer cell metastasis to soft tissues such as the adrenal gland, a documented site for human prostate cancer metastasis. Second, if EMT acquired by prostate cancer cells following cellular interaction with host bone or adrenal gland occurs in patients, this could be a potential target for prevention and treatment strategies. Third, since the host microenvironment was shown to promote EMT and prostate cancer progression, future effort in therapeutic regimens development should be directed at prostate stroma-directed targeting via the use of atrasentan, bisphosphonates, growth factor receptor antagonists, antiangiogenics, and radiopharmaceuticals to effectively eliminate cancer metastases.

Targeting EMT: Potential Clinical Value in Advanced Prostate Cancer One would acknowledge the chanllege to distinguish mesenchymal-like tumor cell from the surrounding mesenchymal cells (stromal cells like fibroblasst and myoblasts) by haematoxylin and eosin staining. Since loss of normal epithelial marker and the variety of the marker expression in those transiting tumor cells, application of the tumor cellular marker immunohistochemistry is also not enough. A set of epithelial and mesenchymal markers immunohistochemistry is used to identify mesenchymal characters and a standard method needs to be established to normalize the evaluation. In order to identify EMT particular morphological features at the invasive front of the tumour away from the tumor mass should be carefully evaluated. A list of proteins implicated in cell– matrix interactions, cell structure and motility (e.g. N-cadherin, Vimentin, Fibronectin, Integrins and FSP-1/S100A4) and secretion of proteolytic enzymes (e.g. MMP2, MMP9) could potentially be used as markers for the identification of cells with mesenchymal characteristics on tumour samples. However, the definition of an EMT, and the requirements to execute one in vitro are at variance with those in vivo and therefore cannot exactly recapitulate these events. It is therefore not surprising to find studies differing in their stringency for the various criteria for defining an EMT.

A variety of extracellular and intracellular signals can trigger transition of epithelial cells to mesenchymal or mesenchymal-like cells during tumor progression.

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TGF-β, EGF family members, FGF, and IGF have all been shown to induce EMT in an autocrine or paracrine manner (Tsuji, Ibaragi et al. 2009). The secretion pattern of these cytokines could be used as supporting evidence of EMT. Transcription factors implicated in the down-regulation of the epithelial E-cadherin for EMT (e.g. SNAIL1, SNAIL2, Twist, EF1/ZEB1 and SIP1/ZEB2) can be analyzed by gene expression assay as potential reliable markers for EMT identification (Voulgari and Pintzas 2009).

As the EMT feature is controlled by multiple cellular signaling, the balance of the signaling network is the key factor to determine the cell fate: epithelial or mesenchymal. With the therapeutical attempt, the manipulation of EMT could be conducted in the context of balancing these cellular signaling events. During tumorgenesis, there is a huge change of microenvironment surrounding the tumor cells (compared to normal tissue) which is one of the potent driving forces for tumor metastasis, as well as EMT. So, manipulating the tumor microenvironment could be a doable strategy by changing the relative ratio of cytokines and growth factors, or applying therapeutic chemicals to the tissue interstitial dynamics. One of the interesting topics is to know whether the widely used taxol chemotherapy drugs could affect EMT towards metastasis. Although the initial analysis did not show significant changes of EMT markers in prostate tissue from the Docetaxel-treated patients, the opportunity for a better study time window and possibly different scheduling of treatment in an expanded patient cohort may reveal some differences. Microtubule targeting drugs can affect AR signaling and functionally interfere with prostate cancer epithelial cell EMT, thus there is an emerging opportunity that microtubule targeting drugs could affect the cell status in terms of EMT.

Microtubules Facilitate AR Nuclear Translocation and Transcriptional Activity Understanding the mechanism drawing therapeutical response to taxanes-based Chemothearpy could result in an increase of clinical benefit in prostate cancer patients is widely accepted recently. Data reported by two independent teams established that a docetaxeil based chemotherapy regimen leads to a significant survival benefit in men with HRPC (Petrylak, Tangen et al. 2004; Tannock, de Wit et al. 2004). Microtubule stabilization through binding of Doxcetaxel to b-tubulin is the most widely accepted

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mdchanism of action. The microtubules cannot disassemble on bound with taxanes, so the static polymerization disrupts the normal mitotic process, arrests the cell in G2M phase, and induce cell death. Another mechanism for the docetaxel is that it functions against the prosurvival effects of Bcl-2 gene expression. Taxol Treatment of prostate cancer cells expressing bcl-2 induces bcl-2 phosphorylation that ultimately inhibits its Bcl-2 binding to bax and apoptosis in taxol-treated prostate cancer cells (Haldar, Chintapalli et al. 1996). The present findings are of major translational significance as they demonstrate a novel mechanism for taxane-based chemotherapy regimens that the inhibition of AR transcriptional activity suppression the prostate cancer cells growth.

Our study shows microtubule targeting chemotherapy drug could inhibit both the androgen dependent and androgen independent activation of androgen receptor by blocking the nuclear translocation of AR. One could speculate that this action potentially provides a complementary blockage of androgen deprivation therapy, an idea that would support combination of ADT and taxanes towards providing additional potent clinical benefit.

Combination of Taxol Chemotherapy with Androgen Deprivation Therapy In the past, prostate cancer was regarded as a “traditionally” chemoresistant malignancy (Eisenberger, Kennedy et al. 1987). However clinical data emerging in the late 1990s, documented the combination efficacy of Mitoxantrone plus either prednisone or hydrocortisone in improving symptoms and quality of life, compared with best supportive care reported in phase III studies (Tannock, Osoba et al. 1996). Thus clinically a “proof of principle” was generated that chemotherapy may result in an increase of clinical benefit for prostate cancer. Mitoxantrone and prednisone were approved as standard treatment by Food and Drug Administration and have been considered as the palliative standard of care for the treatment of patients with symptomatic CRPC since 1999. New evidence from several preclinical and phase I/II demonstrated that microtubule targeting chemotherapy agents suppress tumor growth. (Savarese, Taplin et al. 1999; Oudard, Banu et al. 2005). Subsequent multicenter phase III studies between 1999 and 2000 compared Taxane-based regimens with

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mitoxantrone/prednisone in an attempt to demonstrate improved overall survival compared with standard care. Ongoing studies currently focus on both new promising taxanes and new combination treatments with new drugs. Recent phase II studies have been conducted to determine whether the addition of biological or cytotoxic drugs (Oblimersen, Exisulind, Vinorelbine, Diethylstilbestrol, Bortezomib, Capecitabine, Calcitriol, Bevacizumab, Atrasentan) to docetaxel-based therapy could further improve its efficacy (Mancuso, Oudard et al. 2007).

The AR targeting effect by microtubule targeting drugs documented in this study provides a rationale for a potential combination regimen in the treatment of prostate cancer. In advanced stages of disease, one of the major mechanisms responsible for failure of ADT is the androgen independent activation of AR, shown to promote cell proliferation and metastasis. Microtubule targeting drugs could target the classical AR pathway, as well as the androgen independent AR signaling which might complement ADT. Therefore, combination of microtubule targeting drugs and anti-androgen agents could impair the entire AR signaling which could also be applied to castration-resistant prostate cancer. On the other hand, although the microtubule targeting chemotherapy is reserved only for advanced stage of prostate cancer, the early administration of this chemotherapy with ADT possibly could show their contribution to delay or block the emergence of the castration-resistant stage of prostate cancer as their effect on AR suppression.

Androgens and AR: Key Regulators in Prostate Cancer Progression Androgens and AR are established targets of pharmaceutical intervention for prostate cancer, including treatment with antiandrogens such as bicalutamide and flutamide, which bind to the androgen-binding pocket in the C-terminal ligand-binding domain (LBD) and inhibit hormone-dependent activation of AR. The significant tumor suppression in response to ADT (Huggins and Clark 1940) is often short-lived upon recurrence and progression to advanced disease. Long-term therapy with antiandrogens becomes progressively less effective. Novel strategies to inhibit AR activation, including disruption of ligand- independent AR signaling are needed for developing the next

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generation of prostate cancer treatments modalities. Many studies have been done to understand the underlying mechanism of the emergence of this castration- resistant stage. Several hypothetic mechanisms have been explored: AR gene amplification is found in advanced prostate cancer patients that permits AR overexpression (Visakorpi, Hyytinen et al. 1995). Another mechanism is that mutant AR, which has higher affinity for androgens and could be activated by hypophysical level androgens than wild type AR, is frequently expressed in prostate cancer. Regulation of AR co-factors expression is another mechanism via which AR signaling is enhanced as the induction of AR coactivator and deduction of AR co-suppressor (Dehm and Tindall 2005). In addition, AR crosstalks with diverse growth factor signaling, also contribute to AR activation in castration-resistant prostate cancer stage. Upregulation of growth factors in advanced cancer bypass androgen signaling axis to induce downsteam gene regulation by AR activation. Hypophysical androgen levels after castration could still activate growth factors signaling which can initiate a robust proliferation advantage (Heinlein and Chang 2004).

This work points to a different and perhaps an unappreciated role for AR in advanced prostate cancer. However, there is a paradox defect of this idea: prostate cancer is detected in older men, while it is very rare in young men. As the androgen level decreases in older men, AR expression is also reduced. So, it is an enigma to understand how more prostate cancer occurs in low androgen microenvironment. Our results that AR reversely correlates with androgen induced EMT provides an insight view that low androgen receptor level potentiates the metastatic ability of prostate cancer cells. Therefore, we could ambitiously hypothesize that it is the key for normal growth of prostate epithelial cells to maintain the appropriate threshold level of AR, neither too more or less. By changing its expression level and the help of complicated microenvironment signaling network, AR switches its role between “good cop” and “bad cop” as all the other molecules with double side functions, such as TGFβ.

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Future Directions EMT as a transient step during the process of cancer metastasis, confers the ability of tumor cells, surviving the escape from to original site to migrate and invade to distant sites. Currently, most studies investigating EMT are limited to in vitro evaluation of the phenomenon, since there is a suitable and relevant model is not available model to capture this transient stage in an in vivo setting. As a newly recognized cellular process, EMT, while easily observed on a cellular basis, its overall histopathological evaluation in vivo might be challenging approach. Comparing the biological and molecular differences between the invasive edge and central portion of the tumor in animal model and patient sample is a potential solution. The hormone status of the tumors (e.g., castration-induced androgen deprivation) in these models will provide additional information on the functional contribution of androgen/AR axis to EMT. The expression profile of EMT relevant markers could be evaluated by cDNA microarray and cellular distribution at the histological level could be assessed by tissue microarrays. The approach remains essentially correlative, thus the definition of EMT in the context of tissue architecture in pathological tumor specimens, may still be challenging and potentially misleading. Another possible approach is tracing the tumor epithelial, endothelial and smooth muscle cells in xenograft models of prostate cancer and gathering both the molecular and morphological characteristics of individual cell populations as well as their potential cell- cell interactions. The dissection of the AR function in prostate tumor metastasis is still work in progress by many investigative groups, that is likely to yield new insights into novel approaches for prostate cancer therapy. Evidence generated from experimental in vitro and animal studies presents multiple aspects of AR function; at the clinical setting determining the impact of AR on the outcome of ADT in patients with prostate cancer is the key to draw the real picture of the role AR and androgen axis in prostate cancer progression to castration-resistant disease. Although AR and androgen could play as double edge sword in patients, with early tumor stage, on certain therapeutic regimens and with predictable outcomes, the answer might be as simple as defining a benefit of androgens and AR in patients with CRPC. Determining the function of AR in the response to chemotherapy might also be critical in predicting patient survival and disease

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progression. The combination of microtubule targeting chemotherapy drugs and androgen targeting agents could lead to tremendous clinical benefit in the management of patients with advanced disease. Both the in vitro studies which will determine tumor cell response to the combination treatment and in vivo studies which mimic the clinical outcome must be performed to answer such important questions. Further refined approaches should be considered for characterizing the incidence of EMT in chemotherapy treated prostate cancer specimens. The present work provides the foundation for further exploiting this newly recognized role for AR as a therapeutic platform for targeting specific events initiated not only be androgen-targeting therapeutic modalities, but also by taxol-based chemotherapy towards improved therapeutic benefit in prostate cancer patients.

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Vita

Name

Menglei Zhu

Date of birth

11/01/1980 in Changchun, Jilin, China

EDUCATION

Peking University, 1999 – 2004 M.D., Peking University Health Science Center, People’s Republic of China

University of Kentucky, 2004- now Ph.D. candidate

ACADEMIC EXPERIENCE 2002-2003 Internship, Beijing Hospital, China 2003-2004 Research Assistant, T-cell Research Lab, Department of Immunology, Peking University Health Science Center, China 2004-present Graduate Research Assistant, Graduate Center of Toxicology, University of Kentucky (Mentored by Dr. Natasha Kyprianou)

PUBLICATIONS

1. Fu WX, Zhu ML, Gong SY, Li Y, Chen WF. Molecular cloning and characterization of a novel rat CXC chemokine, rPBP, the homologue of human and mouse PBP. Cytokine. 2004 Apr 7;26(1):37-43. 2. LI Y, Fu WX, Zhu ML, Chen WF. Prokaryotic expression, purification and chemotaxis Analysis of a novel murine CXC chemokine MTCK1/mPBP Chinese Journal of microbiology and Immunology, 2003, 23(6):459-463. 3. Fu WX, Gong SY, Qian XP, Li Y, Zhu ML, Dong XY, Li Y, Chen WF Differential chemotactic potential of mouse platelet basic protein for thymocyte subsets. Cell Mol Life Sci. 2004 Aug;61(15):1935-45. 4. Yu N, Zhu ML, Zhao HB. Prestin is expressed on the whole outer hair cell basolateral surface. Brain Res. 2006 Jun 20; 1095(1):51-8. 5. Yu N, Zhu ML, Johnson B, Liu YP, Jones RO, Zhao HB. Prestin up-regulation in chronic salicylate (aspirin) administration: An implication of functional dependence of prestin expression. Cell Mol Life Sci. 2008 Jun 6. Zhu ML, Partin JV, Bruckheimer EM, Strup SE, Kyprianou N. TGF-βsignaling

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and androgen receptor status determine apoptosis cross-talk in human prostate cancer cell. Prostate. 2008 Feb 15;68(3):287-95. 7. Zhu ML, Kyprianou N. Androgen receptor and Growth factor signaling partnerships in prostate cancer cells. Endocrine-Ralated Cancer. 2008 15: 841-849. 8. Zhu ML, Kyprianou N. Role of androgen and androgen receptor in epithelial- mesenchymal transition of prostate cancer. FASEB J. 2010 Mar;24(3):769-77. 9. Zhu ML, Kyprianou N. Tubulin-Targeting Chemotherapy Impairs Androgen Receptor Activity in Prostate Cancer. (Submitted to Cancer Research)

PRESENTATIONS

1.Zhu, M.L. Kyprianou, N. (2009) Suppression of androgen receptor transcriptional activity by microtubule targeting drugs. Society for Basic Urologic Research annual meeting. New Orleans, LA 2.Zhu, M.L. Kyprianou, N. (2009) Andrgoen induce epithelial mesenchymal transition in prostate cancer. American Association for Cancer Research 100th Annual Meeting. Denver, CO 3.Zhu, M.L. Kyprianou, N.(2008) Androgens and transforming growth factor-β cross-talk to promote prostate cell epithelia mesenchymal transition. Society for Basic Urologic Research annual meeting .Phoenix, AZ 4.Zhu, M.L., Yu, N., Zhao, H.B. (2006) Upregulation of prestin expression in long- term administration of salicylate. The 29th Assoc. Res. Otolaryngol. Annual Meeting. Baltimore, MD 5.Yu, N., Zhu, M.L., Zhao, H.B. (2005) Long-term usage of salicylate upregulates prestin expression in the guinea pig cochlea. The 35rd Society for Neuroscience Annual Meeting. Washington D.C. 6.Fu, W.X., Gong, S.Y., Zhu, M.L., Qian, X.P., Chen, W.F. (2004) Murine PBP (CXCL7) is produced by thymic monocytes/macrophages and preferentially chemoattractant for CD4-CD8- double-negative thymocytes. The 12th International Congress of Immunology. Montreal, Canada.

AWARDS

2004 Research Challenge Trust Fund Fellowships

MEMBERSHIPS

Associate Member of American Association for Cancer Research (2009)

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