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Molecular mechanisms of androgen independence in prostate cancer

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Molecular mechanisms of androgen independence in prostate cancer

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Content MOLECULAR MECHANISMS OF ANDROGEN INDEPENDENCE IN PROSTATE CANCER Copyright 2002 by Yan Shi A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (PATHOBIOLOGY) December 2002 Yan Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 3093968 UMI UMI Microform 3093968 Copyright 2003 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UNIVERSITY OF SOUTHERN CALIFORNIA The Graduate School U niversity Park LOS ANGELES, CALIFORNIA 900894695 This dissertation, w ritten b y Yan S h i U nder th e direction o f A.er. D issertation C om m ittee, and approved b y a ll its m em bers, has been p resen ted to an d accepted b y The G raduate School, in p a rtia l fulfillm ent o f requirem ents fo r th e degree o f DOCTOR OF PHILOSOPHY Dean o f Graduate Studies Date _____ Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DEDICATION To my mom and dad, who inspire me to strive for more with their endless love. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS First of all I wish to thank my mentors, Dr. Clive R. Taylor and Dr. Richard J. Cote for their boundless generosity, perceptive guidance and tremendous input in my dissertation work. I also wish to thank Dr. Ram H. Datar who made a lot of efforts to help me accomplish and present this doctoral work; Dr. Pradip Roy- Burman and Dr. Gerhard Coetzee who gave me a lot of their precious time and valuable suggestions, which are vital to this research project. I wish to thank Dr. Sunanda Chatterjee and Dr. Frank Brands for their excellent work in establishing the clinical cohorts of prostate cancer and initial investigations of differential expressions of proliferative and survival regulators in androgen dependent and independent prostate cancer. I thank Dr. Charles Sawyers for providing me HER 2/neu plasmid; Dr. Zhigang Song for LNCaP cells and Hongwei Li for DHT. I also wish to thank Dr. Dixon Grey and Hal Soucier for their kind help in performing flow cytometry for me. I wish to thank everyone in our lab including Lillian L. Young, Carmela Villajin-Busque, Dongxin Liu, Ben George, Henry Lin, Liana Pootrakul, Dr. Debra Hawes, Dr. Mohammad Alavi, Cheng Liu, William Win, Aiga Charles, Angela McMillian, Janet Aleksanyan, Courtney Conner, Stephen Beil, Raisa Stolitenko and Gina Barles. I would not have been able to complete this work without their support and encouragement. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. I thank Dr. Ashraf Imam who taught me how to do cell culture. I thank Dr. Cheng-Ming Chuong, Dr. Amis Richters and Lisa Doumak for their kind help during all these years. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS Dedication.......................................................................................................................ii Acknowledgements............................................... iii List of T ab les.................................................... ..........................................................vi List of Figures ....... vii Abstract............................................................................ ix Chapter One: Introduction 1.1 Normal prostate gland.............................................................................................. 1 1.2 Prostate cancer ............................................................................................ 1 1.2.1 Epidemiology of prostate cancer 1.2.2 Androgen dependence and androgen independence 1.3 Epidermal growth factor receptor fam ily............................................................. 7 1.3.1 Overview of epidermal growth factor receptor family 1.3.2 HER-2/neu pathway 1.4 PI3K/Akt pathway................................................................................................9 1.4.1 PI3K 1.4.2 Akt 1.4.3 PI3K/Akt 1.5 Cell cycle.................................................................. 11 1.5.1 Overview of cell cycle regulation 1.5.2 Regulation at Gl/S transition 1.6 Apoptosis................................................................. 13 1.7 Purpose of study........................................................................... 14 Chapter Two: Decreased p27 expression is associated with prostate cancer progression 2.1 Introduction................. 21 2.2 Materials and M ethods.............................................................................................21 2.3 Results........................................................................................................................ 25 2.4 Discussion..................... 28 Chapter Three: Coordinate molecular mechanisms in the development of androgen independence in prostate cancer 3.1 Introduction........................................................ 37 3.2 Materials and M ethods............................................................ 40 3.3 Results......................................... 45 3.4 Discussion.............................................................................. 47 v Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter Four: Elevated Her 2/neu expression is associated with prostate cancer progression and androgen independence 4.1 Introduction............................................................................................................... 64 4.2 Materials and M ethods............................................................................................66 4.3 Results........................................................................................................................70 4.4 Discussion..................................................................................................................73 Chapter Five: Her 2/neu overexpression promote androgen independent proliferation and survival 5.1 Introduction............................................................................................................. 85 5.2 Materials and M ethods.............................................................................................88 5.3 Results.........................................................................................................................96 5.4 Discussion..................................................................................................................102 Chapter Six: Conclusions and Perspectives.............................................................. 130 References........................................................................................................................ 133 vi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES Table 2-1: Association of p27 immunoreactivity with tumor grade, stage and PSA levels................................................................................................. 32 Table 3-1: Bcl-2 expression in the untreated, treated and androgen independent group of prostate cancer...................................................................................... 57 Table 3-2: p27 expression in the untreated, treated and androgen independent group of prostate cancer............................................................................................... 59 Table 4-1: Her 2/neu expression in the untreated, treated and androgen independent group of prostate cancer............................................................................ 78 Table 4-2: Association of Her 2/neu expression with Gleason Score and tumor substage in untreated group........................................................................................... 79 Table 4-3: Association of Her 2/neu expression with Gleason Score and tumor substage in treated group ......................................................... 80 Table 5-1: Image analysis data showing similarity of Her 2/neu protein expression in clinical samples compared to HER 2/neu transfected LNCaP cell line m odel ...110 vii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES Figure 1-1: Proposed 5 aberrant pathways of proliferation signaling in androgen independent prostate cancer............................................................................................16 Figure 1-2: PI3K/Akt pathway in cells. ........................................................................18 Figure 1-3: Cell cycle regulation at Gl/S transition.....................................................20 Figure 2-1: Representative p27 immunoreactivity in tissue sections prepared from prostate carcinoma.................................................... 33 Figure 2-2: Association of p27 expression with prognosis of patients with prostate cancer............................................................................................................................... 35 Figure 3-1: Representative pictures of apoptosis detected in the untreated and treated prostate cancer................................................................................................................ 52 Figure 3-2: Apoptotic indices of stage C prostate cancer.......................................... 54 Figure 3-3: Representative immunoreactivity of Bcl-2, p27 and Ki-67 in prostate cancer.............................................................................................................................. 55 Figure 3-4: Bcl-2 expression in the untreated, treated and androgen independent group of prostate cancer................................................................................................58 Figure 3-5: p27 expression in the untreated, treated and androgen independent group of prostate cancer...........................................................................................................60 Figure 3-6: Mean level of Ki-67 expression in the untreated, treated and androgen independent group of prostate cancer.......................................................................... 61 Figure 3-7: Model of coordinate molecular changes following androgen ablation therapy and androgen independence............................................................................62 Figure 4-1: HER-2/neu expression in prostate cancer............................................... 81 Figure 4-2: Association of Fler 2/neu expression with prognosis of prostate cancer patients............................................................................................................................83 Figure 5-1: Her 2/neu status after stable transfection with plasmid containing HER 2/neu cD N A ................................................................................................................... I l l V lll Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 5-2: The growth curves of stably transfected LNCaP cells in culture medium containing FCS or C S S ..................................................................................................113 Figure 5-3: Summary of cell cycle profiles of LN-neo and LN-HER in culture medium containing FCS or C S S .................................................................................. 114 Figure 5-4: p27 expression examined by western blot analysis in the transfected LNCaP cells................................................................................................................... 116 Figure 5-5: Effect of Her 2/neu overexpression on apoptosis...................................119 Figure 5-6: Bcl-2 and Akt expression examined by western blot analysis in transfected LNCaP cells............................................................................................... 121 Figure 5-7: PSA expression examined by western blot analysis in the transfected LNCaP cells................................................................................................................... 123 Figure 5-8: The effect of androgen deprivation and DHT on Her 2/neu expression in transfected LNCAP cells.............................................................................................. 126 Figure 5-9: Model for Her 2/neu overexpression mediated pathway of androgen independence in prostate cancer.................................................................................. 128 ix Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT The primary treatment for advanced prostate cancer is hormone therapy. Nevertheless, resistance to such therapy is a major clinical problem. Several lines of evidence suggest that Her 2/neu overexpression may contribute to the development of androgen independence. In order to further our understanding of development of androgen independence, we established a clinical model system including three cohorts of prostate cancer patients: patients with no hormone therapy, patients who underwent preoperative hormone therapy and patients with advanced tumors resistant to hormone therapy. Immunohistochemical studies of this clinical model system showed that high levels of Her 2/neu and Bcl-2 expression, and low level of p27 expression were significantly associated with the development of androgen independence. Based on these fundamental observations, we postulated that, as a result of androgen deprivation, high level of Her 2/neu expression in androgen independent tumors may provide an alternative survival pathway through up regulating Bcl-2 expression on one hand, and an alternative growth pathway through down regulating p27 expression on the other. In order to test our hypothesis, we stably transfected HER 2/neu gene in androgen responsive LNCaP cells. We found that under androgen-depleted conditions, HER 2/neu transfected cells showed higher proliferation rates, less G1 arrest and lower p27 expression than the mock transfected cells. HER 2/neu transfected cells also showed lower apoptotic rates under hormone- deprived conditions and higher Bcl-2 expression than the controls. Further, There is an elevation of phosphorylated Akt and prostatic specific antigen (PSA) expression X Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. in HER-2/neu transfected cells, which indicates that Her 2/neu overexpression may activate PI3K/Akt signal transduction pathway and androgen receptor (AR) in the absence of androgen. This study elucidates Her 2/neu-mediated mechanism of androgen independence, which functions through: (1) promoting androgen independent survival by upregulating Bcl-2 expression; (2) promoting androgen independent growth by downregulating p27 expression; (3) activation of AR in a ligand independent fashion; (4) activation of the PI3K/Akt signal transduction pathway, through which Her 2/neu promotes androgen independent survival, growth and AR activation. It is hoped that this study may provide new therapeutic strategies for androgen independent prostate cancer. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter One: Introduction 1.1 Normal prostate gland The prostate is a male accessory sex organ surrounding the first portion of the male urethra. It is composed of tubuloalveolar glands arranged in lobules and fibromuscular stroma surrounding the glands. The prostate gland is divided into three distinct zones: peripheral zone, central zone and transitional zone. The peripheral zone is the largest, containing approximately 70% of the prostate volume. It lies posterior and laterally in the prostate. It is the most common site of prostate cancer. The central zone is the part that surrounds the ejaculatory ducts as they course from the base of the prostate to the verumontunum. It contains about 25% of prostate volume. The transitional zone is the part that surrounds the prostatic urethra. It contains about 5% of prostate volume [McNeal 1998]. Under the microscope prostate gland is mainly composed of two types of cells. The luminal glandular cells are columnar or cuboidal, with pale cytoplasm. They secret prostatic specific antigen (PSA) and prostate acid phosphotase (PAP). These cells express androgen receptor and they are androgen dependent. Their function and growth is regulated by androgen, mainly testosterone and dihydrotestosterone (DHT). The basal cells are flat with little cytoplasm. They are arranged at the periphery of prostate glands. These cells do not have secretory function and instead act as stem cells that repopulate the luminal layer [McNeal 1998], 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.2 Prostate cancer 1.2.1 Epidemiology of prostate cancer Prostate cancer is the most commonly diagnosed cancer and also the second leading cause of cancer death in American men [Crum 1994], An estimated 189,000 new cases of prostate cancer were diagnosed in 2002, with 30,200 deaths estimated [www.cancer.org]. It rarely occurs before the age of 40, but the risk rises quickly thereafter [Cram 1994]. With the aging of the population and the improvement of detection methods, the incidence of prostate cancer will continue to rise. It is estimated that one in five American men will develop prostate cancer in their lifetimes. Prostate cancer is not only a major public health problem, but also a huge challenge facing cancer researchers. 1.2.2 Androgen dependence and androgen independence The prostate is an androgen-regulated organ. Androgen is the primary regulator of prostate cell growth, differentiation and function. The effect of androgen on the cell is through androgen signaling pathway. The central element of this pathway in the cell is androgen receptor (AR) that belongs to the superfamily of nuclear receptor. The AR gene is located on the long arm of X chromosome. It encodes a 110-KDa protein consisting of three domains: a C-terminal ligand-binding domain, a central DNA binding domain and an N-terminal transactivation domain. After binding to androgen, AR is activated through a cascade of molecular events including dissociation of heat-shock proteins, phosphorylation, conformational 2 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. changes, dimerization and nuclear translocation. In the nucleus, activated AR binds to hormone response elements (HRE) in the promoters of a variety of androgen regulated genes and transcriptionaly activates these genes [Feldman et al. 2001]. Like the epithelium from which it is derived, prostate carcinoma is an androgen-dependent tumor. As a major growth stimulator and apoptosis inhibitor of prostate cells, androgen plays an important role in the initiation and progression of prostate cancer [Feldman et al. 2001], People with highly undeveloped prostate due to lack of androgen such as constitutional 5a-reductase deficiency do not develop prostate cancer [Ross et al. 1998]. Androgen ablation therapy through orchiectomy induces dramatic regression of androgen dependent prostate cancer as shown by Huggins more than sixty years ago [Huggins 1967], That is why hormonal therapy including androgen ablation and anti-androgen therapy has been the mainstay of therapy for advanced prostate cancer since then. The hormonal management options for androgen deprivation in metastatic prostate cancer include surgical ablation (orchiectomy), or treatment with estrogens, LHRH analogues, and steroidal and non steroidal antiandrogens. Among these, surgical castration or orchiectomy has long been considered the gold standard of endocrine treatment of prostate cancer and has been shown to provide symptomatic relief in 70 - 80% of patients with advanced prostate cancer [Kaisary et al. 1991]. But there are significant side effects associated with this surgical option. The three main pharmaceutical approaches to androgen ablation are administration of exogenous estrogens (such as diethylstilbestrol, ethinyl 3 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. estradiol or premarin), leutinizing hormone-releasing hormone (LHRH) analogues (like leuprolide acetate -Luperon, or goserelin acetate) and combined administration of LHRH-agonists plus anti-androgens (like megestrol, medroxyprogesterone, flutamide and bicalutamide). Estrogen treatments result in androgen blockade by suppressing the pituitary gland, causing the inhibition of LH release, and thereby inhibiting testosterone synthesis and secretion from the Leydig cells in testes. The LHRH analogues first stimulate and then down-regulate the secretion of LH from the anterior pituitary, with a concomitant fall in testosterone and 5-a-DHT. Steroidal antiandrogens have a direct blocking effect at the cellular level and at the level of the hypothalamus. At the hypothalamus, its progesterone-like effects inhibit the gonadotropins LH and FSH, thereby suppressing the testicular production of androgens. [Barradell et al. 1994] However, most of the patients who initially respond to hormonal therapy will eventually develop androgen independent tumor. And there are a few effective therapies so far for these lethal tumors. The molecular mechanisms of androgen independence in prostate cancer remain mostly unknown, and this has been an area of intense investigation in recent years. It is thought that prostate cancer presents as heterogeneous mixtures of androgen dependent and androgen independent cells, with early prostate cancer composed primarily of androgen dependent cells. With androgen ablation therapy, the androgen dependent population undergoes rapid apoptosis or conversion to the androgen independent cell type [Craft et al. 1999]. 4 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The androgen independent cells then dominate the tumor burden. Rapidly proliferating cells may be sensitive to conventional chemotherapeutic drugs, but very slow growth kinetics demonstrated by prostate cancer cells makes them resistant to chemotherapy. Better understanding of molecular mechanisms of androgen independence may lead to the development of new therapeutic strategies for androgen independent prostate cancer. According to our current knowledge, there are five possible pathways leading to androgen independence in prostate cancer (Figure 1-1). The first pathway is “Hypersensitive AR pathway”. This pathway is both androgen and androgen receptor dependent. Even though these tumors seem to be androgen independent, they are actually androgen hypersensitive. They still need androgen, but at much lower levels than normal to stimulate AR pathway. Tumor cells may adapt to the low androgen environment through the following mechanisms: (1) AR amplification, which increases AR content available to androgen in order to maximize the binding between them in presence of androgen deprivation. It has been observed that about 30% of androgen independent tumors that developed after androgen ablation therapy contained amplified AR gene, in contrast none of the tumors from the same patients before such therapy had AR amplification [Koivisto et al. 1997], (2) AR coactivators amplification/overexpression, which enhance AR transactivation at low levels of androgens. A recent study from Gregory et al has 5 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. demonstrated that 5/8 and 6/8 cases of recurrent prostate cancer showed the overexpression of co-activators SRC1 and TIF2, respectively [Gregory et al. 2001]. (3) Increased local androgen level. It has been observed that after androgen ablation therapy, serum testosterone levels are decreased by 95%, but DHT levels in prostate are reduced by only 60% [Labrie et al. 1986]. It is hypothesized that prostate cancer may increase local androgen level through enhanced the activity of 5 a- reductase [Feldman et al. 2001]. (4) Increase AR sensitivity to androgen, which means AR could be activated by low levels of androgen. It may be accomplished by enhancing AR stability and/or nuclear localization [Gregory et al. 2001]. The second pathway is “ Promiscuity pathway”. It is androgen independent but androgen receptor dependent. Tumor cells may adapt to the androgen-deprived environment by mutation, which decreases the specificity of AR and allows non androgen steroids and androgen antagonists to stimulate the AR. One of the best described examples of this kind of mutation is T877A (substitution of alanine for threonine at position 877), which has been found in 25% of metastatic prostate cancer [Gaddipati et al. 1994] and 30% (5/16) tumors treated with flutamide [Taplin et al. 1999]. It is also been found in prostate cancer cell line LNCaP [Veldscholte et al. 1992]. AR with this mutation presents an altered ligand-binding pocket and can be activated by a variety of hormones such as progesterone and estrogens besides androgen. It can also be activated by anti-androgens like flutamide [Veldscholte et 6 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. al. 1992]. Recently, another associated mechanism has been suggested to result in promiscuity by AR; overexpression of AR coactivators increases androgen receptor transactivation at physiological concentrations of adrenal androgen [Gregory et al. 2001]. The third pathway is “ Outlaw pathway”. It is also an androgen independent but androgen receptor dependent pathway. AR is activated by growth factors in ligand-independent fashion. It has been demonstrated that AR could be activated by insulin-like growth factor-1 (IGF-1), keratinocyte growth factor (KGF) and epidermal growth factor (EGF) and interleuking-6 (IL-6) [Culig et al. 1994], probably through cross talk between AR and other growth pathways. Significantly, the AR antagonist casodex can block the activation effects of AR by all of the above [Culig et al. 1994; Feldman et al. 2001]. This indicates that the presence of intact AR is necessary for this kind of mechanism. The fourth pathway is “ bypass pathway”. It is a both androgen and androgen receptor independent pathway. The tumors bypass AR pathway completely. One of the possible bypass mechanisms is through Bcl-2 overexpression. As an apoptosis inhibitor, high Bcl-2 expression protects tumor cells from apoptosis induced by hormone starvation. Thus these tumor cells become androgen independent [Liu et al. 1996]. The fifth pathway is “Lurker cell pathway” [Isaacs 1999]. It is proposed that existence of an androgen-dependent cell population and a small sub-clone of 7 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. hormone-insensitive cells in the primary prostate tumor, with the insensitive clone acquiring a selective growth advantage due to elimination of hormone-sensitive cells by hormone ablation. All these pathways described above may not be mutually exclusive, but rather, they may work together to contribute to development of androgen independence in prostate cancer. 1.3 Epidermal growth factor receptor (EGFR) family 1.3.1 Overview of EGFR family Four members of EGFR family have be identified so far. They are EGFR (erbB- 1 or FIERI), HER2 /neu (erbB-2), erbB-3 (HER3) and erbB-4 (HER4) [Zwick et al. 2001]. We refer to them as the ErbB receptors. The four ErbB receptors have a similar structure consistant with transmembrane receptors, including an extracellular domain, transmembrane domain and cytoplasmic domain with tyrosine kinase activity [Zwick et al. 2001]. They belong to the superfamily of receptor tyrosine kinases (RTK). ErbB receptors are expressed in a variety of epithelial and mesenchymal tissues. They are of great importance in the regulation of cell growth and differentiation. ErbB receptors are activated by a variety of EGF-like peptide growth factors, including EGF, transforming growth factor-a (TGF), heregulin, etc. These growth factors bind specifically to one or several ErbB receptors with different affinities. No 8 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. specific ligand is found for HER2/neu. It is believed that HER2/neu acts as a co receptor in ErbB family [Zwick et al. 2001]. When the ligands bind to the extracellular domain of ErbB receptors, they induce rapid receptor dimerization (either homodimer or heterodimers), with a marked preference for HER2/neu as a dimer partner [Klapper 2000]. Dimerization consequently stimulates the intrinsic tyrosine kinase and autophosphorylation of specific, C-terminal tyrosine residues that provide binding sites for proteins containing Src homology 2 (SH2) or phosphotyrosine binding (PTB) domains. These include a variety of cytoplasmic signaling molecules, such as phosphatidylinositol 3- kinase (PI3K). Through these cytoplamic signaling molecules, the activated ErbB receptors activate intracellular signal transduction pathways, through which regulate the transcription of various downstream genes [Olayioye et al. 2000]. 1.3.2 HER 2/neu HER 2/neu is the most well known member in ErbB family. It was originally identified as both a cDNA clone homologue to EGFR and a transforming gene in a chemically transformed rat neuroblastoma cell line [Coussens et al. 1985; Bargmann et al. 1986; Yamamoto et al. 1986]. Therefore, it got its name as HER 2 (for Human EGF Receptor 2)/neu. Even though no specific ligand for HER 2/neu has been identified, it plays an important role in ErbB family. It is the preferred dimerization partner of all other ErbB receptors, and Her 2/neu containing heterodimers display 9 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. increased ligand affinity due to a decelerated off-rate that can be correlated with prolonged activation of downstream signaling pathways [Zwick et al. 2001]. Overexpression of Her 2/neu has been implicated in numerous types of human tumors, including prostate cancer. The mechanism underlying oncogenic potential of HER2/neu overexpression may due to increased availability for heterodimer formation, which leads to high level of phosphorylation and enhanced cellular signaling through signal transduction pathways including MAP kinase and PI3K/Akt pathway. Therefore, biological responses such as proliferation and survival are enhanced in those cells [Zwick et al. 2001]. In this thesis, we are going to focus on the role of Her 2/neu in the development of androgen independence. 1.4 PI3K/Akt pathway 1.4.1 PI3K phosphatidylinositol 3-kinase (PI3K) is an enzyme that catalyzes the phosphorylation of phosphatidylinositol (Ptdlns), Ptdlns (4) P or Ptdlns (4,5)P2 at the 3’position of the inositol ring to generate Ptdlns (3) P, Ptdlns (3,4)P2 and Ptdlns (3,4,5)P3 [Corvera et al. 1998]. 1.4.2 Akt Akt is discovered because of a unique kind of mice, AKR strain. It has been observed that AKR strain exhibits a high incidence of spontaneous thymoma [Coffer 10 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. et al. 1998]. In 1988, a retrovirus named AKT 8 was isolated from the thymoma cells of the mice [Staal et al. 1988]. This virus was shown to be oncogenic [Staal et al. 1988]. Later in 1991, three groups independently identified and cloned the cellular homologue of AKT 8, which is known as c-Akt 1 or protein kinase B (PKBa) [Bellacosa et al. 1991; Coffer et al. 1991; Jones et al. 1991]. Later, two additional members in Akt family were identified, which were termed as Akt 2/ PKB(3 and Akt 3/PKBy [Datta et al. 1999], The Akt protein is a serine/threonine kinase of 57kDa. It contains multiple domains. The N-terminus contains a pleckstrin homology (PH) domain, which mediates lipid-protein and/or protein-protein interactions important in signal transduction pathway. The central is kinase domain, and the C-terminus contains a hydrophobic and proline-rich regulatory domain [Datta et al. 1999]. It is well known that Akt is important in signal transduction of various cell types. Its kinase activity can be induced by a diverse array of stimulus including growth factors such as platelet derived growth factor (PDGF), basic fibroblast growth factor (bFGF) and EGF [Datta et al. 1999], There are two ways to regulate Akt activity in cells, PI3K dependent and PI3K independent [Datta et al. 1999]. We will focus on PI3K dependent regulation on Akt. 1.4.3 PI3K/Akt pathway Activation of RTK like ErbB receptors induces autophophorylation of specific, C-terminal tyrosine residues through intrinsic tyrosine kinase [Zwick et al. 11 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2001]. The phosphorylated tyrosine residues bind to PI3K and recruit it to the inner surface of the plasma membrane. Once localized to the plasma membrane, PI3K catalyzes the transfer of phosphate from ATP to the 3’ position of the inositol ring of membrane-localized phosphoinositides, thereby generates 3’-phosphorylated phosphoinositides, principally phosphatidylinositol 3,4 biphosphate (PI3,4P) and phosphatidylinositol 3,4,5 triphosphate (PI3,4,5P). These PI3K-generated phospholipids then bind to Akt through its PH domain. The binding results in two changes. One is translocation of Akt from the cytoplasm to the plasma membrane; the other is a conformational alteration of Akt, which makes its Thr-308 and Ser-473 accessible for phosphorylation. In addition, PI3K-generated phospholipids also stimulate 3 -phosphoinositide-dependent protein kinase (PDK) to phosphorylate Akt at Thr-308 and Ser-473, which are two key regulatory sites of Akt. Thr-308 is in the activation loop of the catalytic domain and Ser-473 is in the COOH-terminal domain. Dual phosphorylation at both residues is necessary for full activation of Akt. Activated Akt regulates a variety of intracellular biological responses such as proliferation and survival through phosphorylating a series of downstream proteins [Coffer et al. 1998; Datta et al. 1999]. (Figure 1-2) Akt consensus phosphorylation sequence in vitro has been identified as RXRXXS/T. Using this consensus sequence to search protein database reveals a large number of mammalian proteins that could be Akt substrates. Among these proteins, there are components of cell apoptosis machinery such as BAD, caspase 9, 12 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. apafl [Datta et al. 1999]. Phosphorylation of these death proteins inhibits apoptosis. Therefore, PBK/Akt pathway is a critical survival pathway. 1.5 Cell cycle 1.5.1 Overview of cell cycle regulation It is known that tumors are characterized by abnormal growth. The growth of mammalian cell is regulated through a tight control over the cell cycle. The cell cycle is composed of four sequentially repeated phases: the G1 (the gap before DNA replication), the S (the DNA synthetic phase), the G2 (the gap after DNA replication) and the M (mitotic phase). The progression of cell cycle is triggered by a series of cyclin-dependent kinases (CDKs). The activities of CDKs are tightly controlled by several highly conserved biochemical mechanisms, and regulation through a group of molecules called cyclin-dependent kinase inhibitors (CDIs). There are two families of CDIs, the CIP family which includes p21, p27 and p57, and the INK 4 family which includes pl6, pl5, pl8 and pl9. They bind and inactivate CDK-cyclin complexes [Grana et al. 1995], The cell cycle regulators have been shown to be one of the most common targets of alteration in carcinogenesis, especially those responsible for Gl/S transition [Hartwell et al. 1994], While in G l, mammalian cells integrate the growth- promoting and /or growth-inhibitory signals from the extracellular environment and 13 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. decide either to progress through Gl or enter into quiescence (GO phase) [Grana et al. 1995]. Therefore, Gl phase of the cell cycle is critical for growth factors to regulate cell cycle machinery. 1.5.2 Regulation at Gl/S transition At Gl/S transition, cell cycle progression is controlled by retinoblastoma (Rb) protein and the activity of Rb is regulated through phosphorylation by CDK- cyclin complexes. In Gl arrest, Rb is unphosphorylated and binds to transcription factor (E2F), which prevents the transcription of downstream genes and progression of cell cycle. Meanwhile, CDK-cyclin complexes is inactivated by binding to CDI, such as p27, p21 and p i 6. When cell is stimulated with growth factors, CDK-cyclin complexes is activated by releasing from CDI. Activated CDK-cyclin complexes phosphorylate Rb and release E2F, through which initiate the transcription of downstream genes necessary for cell cycle progression from Gl to S phase [Hartwell et al. 1994; Cordon-Cardo 1995]. p53 is also an important cell cycle regulator at Gl/S transition. Levels of p53 protein increase in response to DNA damage, arresting the cell cycle through increased p21 expression and allowing time for DNA repair [Lane 1992] (Figure 1-3). It is expected that any perturbation along this regulatory pathway at Gl/S transition will lead to uncontrolled cell growth. A better understanding of cell cycle regulation will enable us to explore the molecular mechanism of androgen independent proliferation in prostate cancer. The previous studies including ours suggested that neither p53 nor Rb itself is highly implied in 14 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. prostate cancer progression [Aprikian et al. 1994; Cordon-Cardo 1995; Salem et al. 1997]. Therefore, it is important to assess the potential role of other cell cycle regulators in the process of prostate cancer progression, such as p27. 1.6 Apoptosis Apoptosis is defined as programmed cell death. It is characterized by distinct biochemical and morphological changes, including DNA fragmentation, plasma membrane blebbing and cell shrinkage. In hormone regulated organ like prostate, apoptosis can be induced by hormone withdrawal. Apoptosis is executed by a cascade of sequentially activated caspases. The caspases belong to the family of cysteine proteases that express as inactive pro enzymes in normal healthy cells. When activated, they selectively cleave target proteins at the carboxyl terminus of specific aspartate residues. Caspases cleave both structural proteins and functional proteins. It has been suggested that capases-1, -2, - 8, -9 and -10 are players in the initiation of apoptosis, whereas caspases-3, -6, -7 are in the execution of apoptosis [Datta et al. 1999; Bruckheimer et al. 2000; Kyprianou et al. 2000]. Mitochondria also plays an important role in apoptotic pathway. Several apoptotic stimuli release cytochrome c from the mitochondrial intermembrane space into cytosol where it binds and activates initiator caspase-9. Activated caspase-9 sequentially cleaves and activates executor caspase-3 and -7. Consequently, 15 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. activated caspase-3 and -7 cleave a variety of cellular substrates and generate apoptosis [Datta et al. 1999; Kyprianou et al. 2000]. Since androgen independent prostate cancer is characterized by escape from hormone therapy-induced apoptosis, a better understanding of apoptotic pathway will enable us to explore the molecular mechanism of androgen independent survival in prostate cancer. 1.7 Purpose of study The expansion of prostate cancer depends on the ratio of cells in proliferation to those dying. For androgen dependent tumor, androgen is the main regulator of this ratio by both stimulating proliferation and inhibiting apoptosis. But for androgen independent prostate cancer, alternative molecular mechanisms enable them to survive and proliferate in an androgen depleted environment. Advances in our knowledge of the integrated functions governing prostate cancer proliferation and survival in such condition require the identification of mechanisms underlying these processes. There is growing evidence that androgen independence may develop through activation of alternative growth pathways. One such pathway is the tyrosine kinase pathway mediated by overexpression of the growth factor receptor, Her-2/neu. We and others have recently shown that Her-2/neu overexpression can (a) promote tumor cell proliferation in the absence of androgen and (b) induce anti-apoptosis genes such 16 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. as Bcl-2 and enhance the survival of tumor cells in the absence of androgen [Shi et al, 2001; [Craft et al. 1999; Yeh et al. 1999; Wen et al. 2000], In order to study these issues in detail, we have developed a unique cohort of patient samples representing different stages in the development of androgen independence in prostate cancer; i.e., hormone naive, hormone exposed but sensitive, and hormone-independent prostate cancer. Further, we have developed a cell line model system by which we can directly assess the mechanistic effects of changes in signal transduction pathways. It is our hypothesis that Her-2/neu over-expression is critical to the development of androgen-independence in prostate cancer, specifically through activation of alternative tyrosine kinase growth signaling pathways. We further hypothesize, based on a substantial body of preliminary data, that the PI3 kinase-Akt kinase signaling pathways are activated by Her-2/neu in an androgen deprived environment, thus allowing cell survival and growth in the absence of androgen (i.e., androgen independence). Therefore, we perform this study to explore the molecular mechanisms lead to androgen independence, focusing on Her 2/neu pathway. We believe that a better understanding of this pathway may shed light on the development of new therapeutic strategies for this lethal manifestation of prostate cancer. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 1-1 Proposed 5 aberrant pathways of proliferation-signaling in androgen-independent prostate cancer. a. AR with enhanced sensitivity, either due to higher AR production as a result of AR gene amplification [Visakorpi et al. 1995; Koivisto et al. 1997], enhanced stability and nuclear localization [Gregory et al. 2001] or increased local availability of more potent testesterone to prostate cancer cells [Labrie et al. 1986] b. AR promiscuity due to altered specificity because of gene mutations such as T877A or L701H, allowing binding of non-androgenic ligands flutamide or corticosteroid, or aberrant expression of co-factors of AR such as TIF1 or ARA70 causing broadened AR specificity to allow binding with non-androgens [Miyamoto et al. 1998; Gregory et al. 2001]. c. Outlaw action of steroid hormone receptors allowing ligand-independent AR activation by non-androgenic growth factors (like EGF or IGF) [Sherwood et al. 1998; Kaplan et al. 1999; Nickerson et al. 2001] or RTKs (such as over-expressed Her2/neu) via Akt and/or MAPK pathways [Culig et al. 1994; Craft et al. 1999; Yeh et al. 1999; Wen et al. 2000]. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. d. Androgen-AR bypass through inhibition of apoptosis and promotion of survival by, for example, Bcl-2 over-expression, where altered anti-apoptotic pathways obviate androgen/AR dependence [Liu et al. 1996; Gleave et al. 1999]. e. “Lurker” androgen-insensitive sub-clone of prostate cancer cells, such that the hormone-insensitive clone acquires a selective growth advantage due to elimination of hormone-sensitive cells following hormone ablation. [Isaacs 1999] A Hypersensitive b Promiscuous c Outlaw , ' * > f » •' Gortcof-senxv; t « LufkerceH Adapted and modified from Feldman and Feldman, 2001 19 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 1-2 PI3K/Akt pathway in Cells. Growth factors activation of RTK like ErbB receptors induces autophosphorylation of specific, C-terminal tyrosine residues through intrinsic tyrosine kinase. The phosphorylated tyrosine residues bind to PI3K and recruit it to the inner surface of the plasma membrane. Once localized to the plasma membrane, PI3K catalyzes the transfer of phosphate from ATP to the 3’ position of the inositol ring of membrane-localized phosphoinositides, thereby generates 3 ’-phosphorylated phosphoinositides, principally phosphatidylinositol 3,4 biphosphate (PI3,4P) and phosphatidylinositol 3,4,5 triphosphate (PB,4,5P). These PI3K-generated phospholipids then bind to Akt through its PH domain. The binding results in two changes. One is translocation of Akt from the cytoplasm to the plasma membrane; the other is a conformational alteration of Akt that makes its Thr-308 and Ser-473 accessible for phosphorylation. In addition, PI3K-generated phospholipids also stimulate 3-phosphoinositide-dependent protein kinase (PDK) to phosphorylate Akt at Thr-308 and Ser-473. Activated Akt regulates cell proliferation and survival. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. I Y k v -M i K d i 't o r cell membrane T\rn!ihie k in a M ' Pioiiferation P U k l1 OKI 1 * 1 P J pin l.12>)4U0? P I i- V I lir .M l Sir 47i l’D K l i i \ I ) V ^4 Sur\ival 2 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 1-3. Cell Cycle Regulation at Gl/S Transition. Cell cycle regulation is controlled by protein complexes composed of cyclins (regulatory subunit) and CDKs (catalytic subunit). These complexes phosphorylate and thus inactivate Rb protein at Gl/S transition. Cyclin-CDK complexes are inactivated by binding to CDK inhibitors, such as p27, p21 and pl6. In Gl phase, the unphosphorylated form of Rb protein binds to and sequesters members of the transcription factor family E2F. When phosphlrylated by cyclin and CDK complex, Rb protein releases E2F, which allows the transcription of down stream genes. So the cell cycle can transverse into S phase. p53 regulates cell cycle progression through p21 induction. Y7/ Rb-P Bound E2F Cyclin -cdk Early genes c 2 : Early genes i C l phase p53 S phase 22 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter Two: Decreased p27 expression is associated with prostate cancer progression 2.1 Introduction The management of cancer depends on an accurate assessment of the tumor’s biologic potential. This is particularly true of prostate cancer, now the most commonly diagnosed human cancer. The ability to identify those tumors most likely to progress would greatly facilitate management of the disease. While preoperative serum prostate-specific antigen (PSA) determination has been useful in this regard, it has not shown the specificity for tailoring therapy in individual patients [Partin et al. 1990]. Analysis of genes and proteins involved in cell cycle regulation has yielded important prognostic and treatment information in many tumor systems. Recently, cell cycle inhibitors have been demonstrated to play a potential role in tumor progression [Steeg et al. 1997]. Loss of expression of one of these, the cyclin-kinase inhibitor p27kipl, a member of the cip/kip family [Polyak et al. 1994], has been shown recently to be associated with progression of several tumor types [Loda et al. 1997]. We undertook this study to assess the potential role of p27 expression in prostate cancer progression. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.2 Materials and Methods 2.2.1 Patient population The study included material from ninety six patients (46 - 79 years old; median age = 65 years) with prostate cancer of pathologic stage C (pT3, NO, MO) prostate cancer [1992] who received radical retropubic prostatectomy with bilateral pelvic lymph node dissection at the USC/Norris Comprehensive Cancer Center between 1982 and 1996. Tumors were graded according to the Gleason system [Gleason 1977] and all patients had no evidence of lymph node metastasis as judged by histologic examination. The substage of disease was categorized as described by Gibbons et al [Gibbons 1986]: Cl-invasion through capsule without involvement of surgical margin or seminal vesicles, C2-a positive surgical margin without seminal vesicle involvement, and C3-invovement of seminal vesicle(s). This study was approved by the USC/Norris Institutional Review Board. 2.2.2 Follow-up The median follow-up time for the 96 subjects was 9.5 years. All subjects were evaluated at 1, 2, and 6 months after surgery, followed by 6-month intervals until postoperative year 5, and annually thereafter. Subject evaluation included a complete physical examination and determination of serum prostatic acid phosphatase (PAP) levels and, after 1987, of serum prostatic specific antigen (PSA) levels. Bone scans were performed as clinically indicated or when PSA levels were increased to 0.4 ng/mL or greater. Biopsy specimens were obtained to document 24 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. local cancer recurrence or metastatic disease as clinically indicated. Clinical recurrence of cancer was determined histologically for locally recurrent disease; metastatic disease was confirmed histologically or by bone scan. PSA recurrence was defined as a detectable serum PSA level (0.4 ng/mL) on two consecutive tests determined at least 30 days apart. To determine serum PSA levels, the Hybritech enzyme-linked immuno-absorbent assay was used according to the manufacturer’s instructions (Hybritech, San Diego, CA) for most of the study period; for this assay, “detectable” is defined as a PSA level of 0.4 ng/mL or greater. More recently, a modification of this assay (TOSOH, Foster City, CA) has been used. “Time to clinical recurrence” was defined as the time from prostatectomy to clinical recurrence or until last follow-up, if the subject had not experienced a clinical cancer recurrence: “Time to PSA recurrence” was defined as the time from prostatectomy to the first detectable PSA level or until last follow-up, if the subject had not experienced a PSA recurrence. Participants who died prior to clinical cancer recurrence or a PSA recurrence were censored at the time of death. Survival was calculated as the time from prostatectomy to death due to any cause; patients who are still alive were censored at the date of last contact. 2.2.3 Immunohistochemistry Sections (5-pm-thick) from formalin-fixed, paraffin-embedded primary tumor tissue were mounted on poly-L-lysine-coated slides. Because we found that stored slides gave poor results, all assays were carried out on tissue sections that 25 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. were less than 1 week old from the time of mounting. The slides were deparaffinized with iodine-xylene, washed with absolute ethanol, and rehydrated with 95% ethanol. Endogenous peroxidase was quenched in 3% hydrogen peroxide in absolute methanol. Antigen retrieval was performed as described previously [Shi et al. 1997]. Briefly, samples were processed in citrate buffer (pH6) and heated in a microwave oven twice for 5 minutes (10 minutes in total). After being blocked for nonspecific binding with normal horse serum (20 minutes), tissues were incubated overnight at room temperature with anti-p27 monoclonal antibody (Clone DCS-72.F6; Neomarkers, Fremont, CA) [Rivard et al. 1996] at a dilution of 1:50 in phosphate- buffered saline. The characterization of the p27-specific antibody, including immunochemical detection of cell-overproduced p27 Western blot, has been described [Rivard et al. 1996]. Negative controls consisting of diluents and an irrelevant antibody were used in all experiments. Biotinylated horse anti-mouse secondary antibody (1:200) followed by avidin-biotin conjugate (ABC) (Vector Laboratories, Inc., Burlingame, CA) was applied on the slides. Slides were developed in 0.03% diaminobenzidine as a chromogen, and hematoxylin was used for counterstaining. 2.2.4 Assessment of Immunoreactivity All slides were interpreted by two investigators (R. J. Cote and Y. Shi), who were blinded to all outcome data. The tumors were categorized into one of the following three groups on the basis of the percentage of tumor cell nuclei 26 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. demonstrating p27 immunoreactivity: low p27 expression (0-10%); moderate (11- 50%); and high (>50%). These cutoffs were based on previously published reports [Catzavelos et al. 1997; Loda et al. 1997; Porter et al. 1997] demonstrating the prognostic significance of p27 expression in colon and breast carcinomas. In all cases, tissue sections for the entire prostatectomy specimen were reviewed, and tissue blocks containing the most representative areas of the tumor were selected for assessment. The level of p27 immunoreacivity was determined after the entire tissue section was examined at 40x and lOOx power (Olympus BH- microscope, Melville, NY). Heterogeneity of p27 immunoreactivity in the tumor was reflected in the score and represents an overall assessment of the entire tissue section. Normal prostate glands in the tissue sections were used as internal positive controls to monitor the quality of the immunohistochemical reaction. 2.2.5 Statistical Analysis The Mantel-Haenszel test for trend [Mantel 1963] was used to evaluate the association between p27 immunoreactivity (low versus moderate versus high) and tumor grade (Gleason’s score 4 - 6 versus 7 -10), tumor substage (Cl versus C2 versus C3), and PSA level obtained before surgery (divided into three groups: < 4.0ng/mL versus 4 - 1 0 ng/mL versus >10 ng/mL), measured prior to any biopsy or surgical procedure involving the prostate. Kaplan-Meier plots [Kaplan et al. 1958] and the logrank test [Miller 1981] were use to assess the association of p27 levels with survival, time to clinical recurrence, and PSA recurrence; standard errors were 27 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. based on Greenwood’s formula [Miller 1981]. The stratified logrank test was used to determine whether p27 expression provided prognostic information beyond Gleason score and tumor substage [Breslow 1988]. All reported p values are two-sided. Hazard ratios were used to calculate the relative risks of death or recurrence (clinical and/or PSA). These calculations were based on the pike estimate using the observed and expected number of events as calculated in the logrank test statistic [Pike 1972]; 95% confidence intervals (CIs) used variances derived from the information matrix [Lawless 1982; Berry et al. 1991]. 2.3 Results 2.3.1 Immunoreactivity of p27 in normal and non-malignant prostate glands Nuclear reactivity for p27 was seen in the luminal and basal cells of the normal prostate glands in all cases, which served as internal controls for the assessment of p27 expression. The immunoreactivity of p27 was also observed in the basal cells of hyperplastic and prostate carcinoma in situ (PIN), but not in the luminal cells. These findings of p27 expression are consistant with the other reports [Cordon-Cardo et al. 1998]. 2.3.2 Immunoreactivity of p27 in stage C prostate cancer Of the 96 stage C prostate cancer we examined, 53 (55%) showed a high level of p27 expression, 31 (32%) showed a moderate level, and 12 (13%) showed a low or undetectable level (Figure 2-1). 28 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.3.3 Association of p27 expression with intermediate prognostic markers of prostate cancer To perform an initial assessment of the potential clinical utility of p27 in prostate cancer, we determined the association of p27 immunoreactivity with known intermediate markers of prostate cancer progression, i.e., Gleason score, tumor substage (Cl, C2 or C3), and preoperative serum PSA levels. There was a significant association between p27 immunoreactivity and Gleason score (p = 0.004); tumors with a higher Gleason score tended to have decreased expression of p27 (Table 2-1). However, the level of p27 immunoreactivity was not significantly associated with tumor substage (p = 0.320) or preoperative serum PSA for the 37 patients who had this test performed (p = 0.360) (Table 2-1). Thus, p27 expression does not appear to be related to tumor volume, at least as assessed by preoperative PSA levels, although the sample size for this analysis was small. 2.3.4 Association of p27 expression with recurrence and survival To assess further the value of p27 expression as a prognostic marker of prostate cancer, we evaluated the association of p27 immunoreactivity with overall survival and recurrence risk. We determined that decreased p27 immunoreactivity was significantly associated with an increased probability of recurrence (clinical and/or PSA) (p = 0.004) and decreased survival (p = 0.010) (Figure 2-2A and B). The probability of a subject in this study with high versus moderate versus low p27 immunoreactive tumors remaining recurrence free after 9 years was 67% (95% Cl = 29 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 54 - 80%) versus 47% (95% CI=29 - 65%) versus 17% (95% Cl = 0 - 43%), respectively. Similarly, the probability of remaining alive after 9 years was 92% (95% Cl = 85 - 99%) versus 79% (95% Cl = 64 -94%) versus 49% (95% Cl = 20 - 78%), respectively. The median recurrence-free interval for those subjects with tumors demonstrating high versus moderate versus low p27 immunoreactivity was 13.7 years versus 8.4 years versus 4.7 years, respectively. The median survival was more than 14 years versus more than 13.5 years versus 8.1 years, respectively, for the high versus moderate versus low p27 immunoreactivity groups. The relative risk of recurrence for patients with tumors demonstrating high versus moderate versus low p27 immunoreactivity was 1.00 versus 2.00 (95% Cl = 1.10 - 3.60) versus 3.31 (95% Cl = 1.61 - 6.73), respectively. The relative risk of dying was 1.00 versus 1.69 (95% Cl = 0.67 - 4.25) versus 8.24 (95% Cl = 1.79 - 12.30), respectively. A multivariable analysis of the survival data demonstrated that p27 status was independent of tumor grade and substage in predicting survival (p = 0.040). Of particular interest, we observed few deaths before 6 years after surgery, and substantial differences in survival were only noted beginning 8 years from surgery. (Figure 2-2 A) 2.4 Discussion As a result of more widespread use of sensitive screening methods for prostate cancer, in particular serum PSA levels, the number of diagnosed prostate 30 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cancers has increased dramatically in recent years. Despite the increased detection, many of these cancers would have likely remained clinically occult without any treatment [Johansson et al. 1992]. The larger issue is prognosis, where assessment of prostate cancer has been limited to the identification of tumor grade, tumor stage and volume, and pre-operative PSA levels [Partin et al. 1990]. These measures, while powerful, cannot identify the specific individuals most likely to develop metastatic prostate cancer, particularly those patients with clinically localized disease (i.e., those patients for whom prognostic analysis is most important in management). Thus, there is a great need to define more specific and sensitive ways to assess the biologic potential of prostate cancer. Identifying those patients most likely to experience tumor progression is of obvious medical and economic importance. Our results support the idea that the level of p27 expression is a predictor of recurrence and survival in patients with stage C prostate carcinoma. Subjects in this study with tumors that maintained high levels of p27 immunoreactivity had significantly longer disease-free intervals and longer survival than did subjects with low p27 expression. Those men with tumors showing moderate levels of p27 immunoreactivity had a probability of recurrence and survival that were intermediate, compared with the high and low p27 immunoreactivity groups. Furthermore, the majority of patients showed high levels of p27 expression; these patients tended to remain recurrence free and alive for long periods. Thus, p27 is an independent and significant predictor of prostate cancer progression. 31 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The role of cell cycle regulation in prostate cancer progression is not unexpected; perturbation in the cell cycle has been associated with prognosis for many tumors [Esrig et al. 1994; Cordon-Cardo 1995]. However, to date, the most obvious candidate, p53, has not been shown to play a major role in prostate cancer tumorigenesis or progression [Aprikian et al. 1994; Grignon et al. 1997; Salem et al. 1997]. We undertook this study to assess the potential importance of another pathway in cell cycle regulation and prostate cancer progression. p27 is a member of the kip/cip family of cyclin-dependent kinase inhibitors and is involved in regulation of the cell cycle at the Gl/S transition. It inhibits the kinase activity of cyclin E- CDK2 complex that is necessary for retinoblastoma protein phosphorylation [Cordon-Cardo 1995]. Through keeping Rb in unphosphorylated condition, p27 stops cell cycle progression at Gl/S transition. While p27-deficient mice display organomegaly, they do not form cancers [Fero et al. 1996; Kiyokawa et al. 1996; Nakayama et al. 1996]. But loss of p27 expression has been shown to be associated with progression of several tumor types, including colon and breast cancer [Catzavelos et al. 1997; Loda et al. 1997; Porter et al. 1997]. These studies suggest that the loss of p27 contributes the progression of at least a subset of tumors, probably through uncontrolled cell growth. Even though loss of p27 expression has been shown to be common in many cancer cells, mutations in the human p27 gene appear to be rare [Ponce-Castaneda et al. 1995; Ferr and o et al. 1996]. It has been found that the loss of p27 expression 32 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. appears to be due to increased proteasome-mediated protein degradation and not to p27 gene alteration or messenger (mRNA) instability [Loda et al. 1997], These studies are important for two reasons: 1) They implicate the p27 pathway in progression of at least a subset of tumors, and 2) they demonstrate that p27 analysis must take place at the level of the protein. Thus, immunohistochemical method becomes the tool of choice for determining the p27 status of tumor cells. Altered p27 gene expression does appear to play a role in the development of benign prostate glandular hyperplasia in humans; decreased p27 mRNA expression is associated with pro static epithelial hyperplasia [Cordon-Cardo et al. 1998]. Thus, loss of p27 expression appears to play a role in both prostate tumorigenesis and progression as well as in the development of benign epithelial hyperplasia, but through two distinct pathways. The patient population included in this study is unique in that it is made up of individuals who received the same surgical treatment (radical prostatectomy with pelvic lymph node dissection), who had a consistent pathologic stage of disease, who were well characterized with regard to tumor grade, and who had long-term (median 9.5 years) follow-up. Thus, many of the confounding variables typically present in studies involving prostate cancer (most notably differences in surgical treatment and stage of disease) were not present in this population. Patients with stage C disease have clinically localized cancer (i.e., no evidence of lymph node or systemic metastasis), but they have higher recurrence rates than patients with pathologic stage 33 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. B disease (who have extremely low rates of prostate cancer recurrence). Thus, studies involving patients with stage C disease have a grater power in assessing biologic differences in tumors. Finally, because of the natural history of the disease, studies evaluation clinical outcome in prostate cancer require a long follow-up. An assessment of p27 expression may be important in determining the biologic potential of prostate carcinoma. The interaction of p27 with other cell cycle regulatory proteins (such as p21 w af 1/cipl) and the role of p27 in the induction of androgen and chemotherapy resistance [St Croix et al. 1996] are the topics of ongoing research and will help to better define how p27 expression should be used in managing patients with prostate cancer. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 2-1 Association of p27 immunoreacivity with tumor grade, stage, and preoperative prostate-specific antigen (PSA) levels. Immunochemical detection of p27 was performed by use of 5-pm-thick sections of tumor tissue and a monoclonal p27 antibody. Classification of tumors into low, moderate, and high p27 immunoreactivity groups was based on the percentage of tumor cell nuclei demonstrating p27 immunoreactivity: low p27 expression (0 - 10%), moderate p27 expression (11 - 50%) and high p27 expression (> 50%). P27 immunoreactivity, % of patients No. of ---------------------------------------------- subjects Low Moderate High All subjects 96 13 32 55 Gleason’s score 4-6 36 3 25 72 7-10 60 18 37 45 Tumor substage Cl 32 9 34 56 C2 33 12 24 64 C3 31 16 39 45 Preoperative serum PSA <4.0 ng/mL 10 0 30 70 4-10 ng/mL 11 0 27 73 >10 ng/mL 16 12 25 62 0.004 0.320 0.360 35 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2-1: Representative p27 immunoreactivity in tissue sections prepared from prostate carcinoma. A) Tumor demonstrating high p27 expression (> 50% of tumor cell nuclei show brown color). B) Tumor demonstrating moderate p27 expression (11 - 50% of tumor cell nuclei). C) Tumor demonstrating low p27 expression (0 - 10% of tumor cell nuclei). Note the adjacent normal prostate gland tissue with p27 nuclear immunoreactivity, used as an internal control (arrow). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. B 37 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2-2 Association of p27 expression with prognosis of patients with prostate cancer. A. Probability of remaining recurrence free in 96 patients with stage C prostate cancer, based on the level of p27 immunoreactivity. The p value was based on the logrank test. Each tick mark represents a patient who had no evidence of disease at the last follow-up visit. The patients with tumors showing high p27 expression had highest probability of remaining recurrence free, while the patients with tumors showing low p27 expression have lowest probability of remaining recurrence free. The probability of a subject in this study with high versus moderate versus low p27 immunoreactive tumors remaining recurrence free after 9 years was 67% (95% Cl = 54 - 80%) versus 47% (95% CI=29 - 65%) versus 17% (95% CI = 0 - 43%), respectively. B. Probability of survival in 96 patients with stage C prostate cancer, based on the level of p27 immunoreactivity. The p value was based on the logrank test. Each tick mark represents a patient who was alive at the last follow-up visit. The patients with high p27 expression had highest probability of survival, while the patients with low p27 expression have lowest probability of survival. The probability of remaining alive after 9 years was 92% (95% Cl = 85 - 99%) versus 79% (95% Cl - 64 -94%) versus 49% (95% Cl = 20 - 78%), respectively. 38 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. Estim ated Probability of Surviving 8 8 O o o o o o o o o o > o o ’a 'o 3 3 * :c Estim ated Probability of Not Recurring £ o 3 o o , c to CD * 5 8 8 o o o o o o o o Chapter Three: Coordinate molecular mechanisms in the development of androgen independence in prostate cancer 3.1 Introduction Carcinoma of the prostate is the most common malignancy in men and is the second leading cause of death due to cancer. Unfortunately, a lot of patients with prostate cancer present with advanced disease. The primary treatment for metastatic prostate cancer is androgen ablation and anti-androgen therapy. Nevertheless, resistance to hormone therapy is a major clinical problem in prostate cancer. Most, but not all patients show initial response to such therapy, but almost all patients who do respond initially will eventually develop androgen independent disease [Sinha et al. 1977], Despite advances in anti-cancer therapy, there is no effective cure for androgen independent prostate cancer. The median survival in such cases is only 9- 12 months [Henry 1999]. The molecular mechanisms responsible for androgen independence are not clear. Knowledge of molecular changes caused by hormone therapy and the differential expression of molecular markers in androgen dependent and independent cancer may lead to a better understanding of the events resulting in androgen independence. We have established a unique cohort of patient with prostate cancer that include patients without exposure to hormone therapy, patients with pre operative exposure to hormone therapy and patients with metastatic androgen 40 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. resistant prostate cancer. These cohorts represent different stages of development along the pathway of androgen independence. These three cohorts were designed to represent different development stages of androgen independence from androgen dependent and not treated with hormone therapy, through treated but still androgen dependent to therapy resistant and androgen independent prostate cancer. Studies of differential gene expression among these three groups may shed light on our understanding of changes leading to androgen independence. It is clear that the development of androgen independence is a multi-step process. The tumor cells need to establish alternative growth and survival mechanisms in the androgen deprivation environment. The purpose of this study was to elucidate the molecular mechanisms of androgen independence through examining both survival and growth regulators in the three cohorts of prostate cancer. As we have mentioned in Chapter 1, androgen is the main regulator of survival for androgen dependent prostate cancer. Androgen withdrawal through hormone therapy induces massive cell death (apoptosis) in the tumors [Huggins 1967]. But unfortunately, many of the androgen dependent prostate cancer eventually find a way to escape hormone therapy-induced apoptosis and become androgen independent. Bcl-2 protein is a well-known apoptosis inhibitor. It was discovered as a proto-oncogene associated with the t(14;18) chromosomal translocation in follicular lymphoma [Tsujimoto et al. 1986]. Bcl-2 gene is located on chromosome 18q21. It encodes a 26 kD protein which has been found in nuclear 41 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. envelope, endoplasmic reticulum and outer mitochondrial membranes [Krajewski et al. 1993]. Forced Bcl-2 expression resulted in suppression of apoptotic cell death, rather than stimulation of cell growth [Kyprianou et al. 2000]. In the prostate, Bcl-2 expression is usually restricted to the basal cells of but not the secretory epithelial cells of prostate glands. Therefore, the basal cells are resistant to androgen ablation, but the secretory epithelial cells are not. They undergo apoptosis following androgen ablation [McDonnell et al. 1992; Colombel et al. 1993]. It also has been shown that Bcl-2 expression in prostate is augmented by androgen ablation. Bcl-2 overexpressed in prostate cancers is associated with clinically androgen independent tumor [McDonnell et al. 1992; Colombel et al. 1993]. Our cohorts of patients are ideal to study the role of Bcl-2 in the development of androgen independence, with the pre-operatively treated group representing the early events, and the androgen independent group representing the late events that occur after long-term hormonal therapy. We expect the difference of Bcl-2 expression among the three cohorts of prostate cancer may provide us more information on survival part of androgen independent pathway. Proliferation is another important part of androgen independent pathway. Cell growth is tightly controlled under a series of cell cycle regulators. As a negative cell cycle regulator, p27 works at Gl/S transition. We have previously shown that it is an important predictor of prostate cancer progression [Cote et al. 1998]. Low levels of p27 are strongly associated with high Gleason grade (Gleason grade 7-10), and 42 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. patients with prostate cancers expressing low p27 have higher risk of recurrence and lower survival rates [Cote et al. 1998]. It has been shown that the level and/or activity of p27 is decreased by serum mitogens, growth factors, and increased by TGF-beta and cell-cell contact [Polyak et al. 1994], Androgen-sensitive LNCaP cells have been found expressing a high level of p27K IP 1 after androgen deprivation [Knudsen et al. 1998]. We expect the difference of p27 expression along with the expression of proliferative protein Ki-67 among the three cohorts of prostate cancer may provide us more information on proliferative dysregulation associated with androgen independence. 3.2 Materials and Methods 3.2.1 Patient Population The study included material from eighty-one patients with prostate cancer, including sixty-one cases of pathologic stage C (pT3, NO, MO) prostate cancer [1992] who [1992] received radical retropubic prostatectomy with bilateral pelvic lymph node dissection at the USC/Norris Comprehensive Cancer Center between 1982 and 1996, and 20 cases of advanced prostate cancer who had rising prostate specific antigen (PSA) despite hormonal ablation via orchiectomy and systemic hormone therapy. These patients underwent transurethral resection for the relief of urinary obstruction at Ruhr University, Bochum, Germany between 1990 and 1992. According to their treatment status, patients were subdivided into the following groups: (1) Untreated group, (46-79 years, median age = 65 years): tumors from 43 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. thirty-one patients with pathologic stage C disease who had no exposure to androgen ablation therapy prior to radical prostatectomy (2) Treated group (54-77 years, median = 66 years): tumors from thirty patients with pathologic stage C disease who received neoadjuvant androgen ablation therapy (Diethylstilbestrol, lmg twice or four times per day) prior to radical prostatectomy. The duration of DES treatment varied from 3 days to 20 weeks. This group was hormone treated, but considered to be androgen responsive. (3) Androgen independent group (50-91 years old, median age = 74 years): tumors from twenty patients with advanced, androgen independent prostate cancer that had rising PSA despite orchiectomy and systemic anti-androgen or androgen ablation therapy (Fifteen patients had cyproterone acetate 50-150 mg daily. The duration varied from 2 to 96 months. Another two patients had received LHRH analogue for 2 years, 3.6 mg per month. The remaining two patients had orchiectomy but did not have any systemic hormone therapy). All tumors were graded according to the Gleason system [Gleason 1977]. Gleason scores were matched between the untreated and treated group. The substage of disease was categorized as described by Gibbons et al [Gibbons 1986]: Cl- invasion through capsule without involvement of surgical margin or seminal vesicles, C2- a positive surgical margin without seminal vesicle involvement, and C3-involvement of seminal vesicle(s). Because seminal vesicle invasion usually confers a worse outcome, we stratified the 61 cases of stage C prostate cancer into two groups: C1/C2 vs. C3. 44 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.2.2 Detection of Apoptotic Cells Using The In Situ End-Labeling Method Formalin-fixed, paraffin-embedded tissue slides were deparaffinized and treated with protease for 5 minutes at 37° C. The slides were then incubated with digoxigenin-labeled dUTP, dATP and the enzyme terminal deoxy-nucleotidyl transferase (TdT), according to the manufacturer's protocol (ApopTag Kit, Oncor, MD). TdT catalyzes template-independent addition of nucleotides to the free 3'-OH ends of DNA fragments that are present in the nuclei of cells undergoing apoptosis. Normal cells do not contain significant amounts of DNA fragments with free 3'-OH ends and are therefore not labeled by this technique. The slides were then incubated with peroxidase-conjugated anti-digoxigenin antibody at room temperature for 30 minutes. After washing thoroughly with PBS, freshly prepared diaminobenzidine (DAB) was used as the chromagen and the slides subsequently counterstained with hematoxylin. Apoptotic cells (i.e., those cells with labeled 3'-OH ends) were identified by the brown color of their nuclei. In addition, positively stained cells were confirmed using morphologic criteria for apoptosis (chromatin condensation, cell membrane blebbing, and apoptotic bodies). Sections of human tonsil, which demonstrate apoptotic cells in the germinal centers, were used as positive controls. The specificity of the method was confirmed by substituting PBS for the enzyme TdT for negative controls. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.2.3 Quantification of The Apoptotic Cells Apoptotic tumor cells were identified by the brown color of their nuclei and confirmed using morphological criteria, as described above. In each case, positively stained tumor cells were counted from ten contiguous high power fields (HPF) containing tumor. Prior studies have shown that a cell undergoing apoptosis can appear as a cluster of apoptotic bodies [Aihara et al. 1994]. Thus, when an occasional cluster of apoptotic bodies was observed, this was counted as one cell. Because the relatively few apoptotic cells found in the prostatic duct lumen are likely to persist for an indefinite period of time, they were not scored [Aihara et al. 1994]. The level of apoptosis for each case was expressed as the Apoptotic Index, defined as the number of apoptotic cells detected per 100 tumor cells [Kasagi et al. 1994], 3.2.4 Immunohistochemistry Formalin-fixed paraffm-embeded tissues were cut into 5 pm thick serial sections and placed on Probe-On Plus slides (Fisher Scientific, Pittsburgh, Pennsylvania). After deparaffinization in xylene for ten minutes, the slides were dehydrated with absolute ethanol, and then rehydrated with 95% ethanol. Endogenous peroxidase was quenched in 3% hydrogen peroxide in absolute methanol. Antigen retrieval was performed as described previously [Shi et al. 1997]. Briefly, the slides were soaked in citrate buffer (pH6) and heated in a microwave oven twice for 5 minutes (10 minutes in total). After being blocked for nonspecific binding with normal horse serum (20 minutes) at room temperature, tissues were 46 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. incubated overnight at room temperature with primary monoclonal antibodies: p27 (NeoMarkers, Fremont, CA) at a dilution of 1:50; Ki-67 (Mib-1, Vector Laboratories, Inc., Burlingame, CA) at a dilution of 1:50; and Bcl-2 (Dako, Carpinteria, CA) at a dilution of 1:20 in phosphate-buffered saline. Biotinylated horse anti-mouse secondary antibody (1:200) followed by avidin-biotin conjugate (ABC) (Vector Laboratories, Inc., Burlingame, CA) was applied on the slides. Slides were developed in 0.03% diaminobenzidine as a chromogen, and hematoxylin was used for counterstaining. Reactive lymph nodes were used as a positive control for Bcl-2 and Ki-67, and known positive cases of prostate cancer were used as controls for p27 and androgen receptor. 3.2.5 Assessment of Immunoreactivity In all cases, tissue sections for the entire prostatectomy specimen were reviewed, and tissue blocks containing the most representative areas of the tumor were selected for assessment. The level of immunoreacivity was determined after the entire tissue section was examined at 40x and lOOx power (Olympus BH- microscope, Melville, NY). Heterogeneity of the immunoreactivity in the tumor was reflected in the score and represents an overall assessment of the entire tissue section. All slides of the untreated and treated group were interpreted by Y. Shi and S. Chatterjee, and the slides of the androgen independent group were reviewed by Y. 47 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Shi and F. Brands independently. The discrepant cases were re-assessed until concordance was reached. The nuclear immunoreactivity of p27 was observed in the epithelial cells of normal prostate glands, which served as internal positive controls to monitor the quality of the immunohistochemical reaction in each case. The tumors were categorized into one of the following two groups on the basis of the percentage of tumor cell nuclei demonstrating p27 immunoreactivity: low p27 expression (0 - 10%); and high (>10%). The cytoplasmic immunoreactivity of Bcl-2 was observed in lymphocytes, which served as internal positive controls to monitor the quality of immunohistochemical reaction in each case. The tumors were categorized into one of the following two groups on the basis of the percentage of tumor cell cytoplasm demonstrating Bcl-2 immunoreactivity: low Bcl-2 expression (0 - 10%) and high (> 10%). The nuclear immunoreactivity of Ki-67 was regarded as positive. The tumor cells showing positive Ki-67 immunoreactivity were counted under X400 high power field. For each case, we count ten high power fields. The average number of positive cells from ten high power fields was used as the score for each case. 3.2.6 Statistical Analysis The student t test was used to compare the mean apoptotic indices and mean level of Ki-67 expression among the untreated, treated and androgen independent 48 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. group. The chi-square test was used to compare the number of patients with high or low apoptotic indices, high or low expression of p27 and Bcl-2 among the untreated, treated and androgen independent group. The results were considered significant if the two-sided p value was less than 0.05. 3.3 Results 3.3.1 Detection of apoptotic cells Apoptotic cells were easily identified based on nuclear staining (Figure 3-1). As has been observed by other groups [Kasagi et al. 1994], some of the samples demonstrated moderately positive signals in normal non-apoptotic nuclei of tumor cells. However, even in these cases, the signals from the nuclei of cells demonstrating the morphologic characteristics of apoptotic cells were uniformly intense and not difficult to distinguish from the background staining. 3.3.2 Comparison of the apoptotic indices between untreated and hormone treated groups The mean apoptotic index of the hormone treated prostate cancers was significantly higher than that for the untreated cancers (mean=2.25 vs 0.82 respectively, p<0.001). The median apoptotic index from the 31 untreated cases was 0.75. This value was used as a 'baseline' value of apoptotic indices for pathologic stage C prostate cancer and was then used to compare the apoptotic indices between the hormone treated group and the control group. The number of patients that 49 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. demonstrated apoptotic indices greater than the median of 0.75 was significantly higher in the hormone treated group (26 of 30 cases, 87%) compared to the untreated group (14 of 31 cases, 45.1%, p=0.025) (Figure 3-2). Four out of 30 treated cases (13%) demonstrated apoptotic indices lower than the baseline apoptotic index of 0.75. Because of the fixation issue, the apoptotic indices of androgen independent prostate cancer were not available for this study. 3.3.3 Expression of Bcl-2 in prostate cancer Bcl-2 cytoplasmic immunoreactivity was observed in the basal but not in the luminal cells of normal prostate glands, which serves as internal controls for the assessment of Bcl-2 expression (Figure 3-3). The finding of Bcl-2 expression in prostate basal cells is consistent with the previous reports [McDonnell et al. 1992; Colombel et al. 1993]. Bcl-2 expression was generally low in the untreated prostate cancers. Of 22 cases in the untreated group, only 2 (9%) showed high Bcl-2 expression. In contrast, 15 of the 30 cases (50%) showed high Bcl-2 expression in the treated group. The difference between these two groups was highly significant (p = 0.002, Table 3-1, Figure 3-4). Of 28 cases of androgen independent prostate cancer, 20 (71%) showed high Bcl-2 expression (Table 3-1, Figure 3-4). Compared to the untreated and treated group, Bcl-2 expression was significantly elevated in androgen independent group (p < 0.001). The difference in Bcl-2 expression was still significant when compared 50 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. separately with the untreated group (p < 0.001), but not with the treated group (p = 0.096). 3.3.4 p27 expression in prostate cancers Of 31 cases in the untreated group, 13 (42%) showed high p27 expression (Table 3-2, Figure 3-3 and 3-5). In contrast, 21 of the 29 cases (72%) in the treated group showed high p27 expression (Table 3-2, Figure 3-5). The difference between these two groups was statistical significant (p = 0.04). The androgen independent group showed the lowest p27 expression among these three groups. Of 26 cases in androgen independent group, only 4 (15%) showed high p27 expression (Table 3-2, Figure 3-5). Compared to the untreated and treated group, p27 expression was significantly decreased in androgen independent prostate cancer (p < 0.001). The difference in p27 expression was still significant when compared separately with the untreated and treated groups (p = 0.04, and < 0.001, respectively). 3.3.5 Ki-67 expression in prostate cancers To further assess the proliferation ability of these three groups of prostate cancer, we examined Ki-67 expression (Figure 3-3). A mean value was calculated for each of the three groups; mean Ki-67 expression was found to be 5.3% in the untreated, versus 5.5% in the treated, versus 19.2% in the androgen independent groups. The expression of Ki-67 was dramatically increased in androgen independent 51 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. group. The difference of Ki-67 expression among the three groups was significant (p = 0.004) (Figure 3-6). 3.4 Discussion Using the three cohorts of prostate cancer patients which includes those never exposed to hormone therapy, exposed but sensitive to hormone therapy, and exposed but resistant to hormone therapy, we have investigated the coordinate molecular changes of both survival and proliferation aspects during the development of androgen independence of prostate cancer. It has been suggested that in the absence of survival signals (such as growth factors) certain cells are induced to undergo apoptosis [Thompson 1995]. In the case of prostatic epithelial cells, the physiologic growth stimulus is the presence of androgens. We have demonstrated that prostate cancers treated with preoperative hormonal ablation therapy show evidence of increased cell death in the form of apoptosis. Because both the treated and untreated (control) groups were matched with respect to stage, Gleason score, and age, these results indicate that the increased cell death observed in the treated tumors was a direct consequence of androgen withdrawal. In this study, we have observed progressive elevation in Bcl-2 expression from androgen sensitive untreated cancer through androgen sensitive treated cancer to androgen independent cancer (Figure 3-2). High Bcl-2 expression is common in 52 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. androgen independent prostate cancer and it is significantly associated with the development of androgen independence (p < 0.001). This finding is consistent with the previous report of McDonnell et al. They found that Bcl-2 was undetectable in most of their androgen-dependent cases (13/19), but was displayed at high level in almost all of their androgen independent cases [McDonnell et al. 1992], These results imply that overexprssion of apoptosis-supprissing proto-oncogene Bcl-2 may be one of the reasons that androgen independent cancer can survive in an androgen- deprived environment. Our study has also found that Bcl-2 expression highly associated with initial hormonal ablation therapy (p = 0.002), suggesting that Bcl-2 overexpression is induced by hormonal ablation therapy. McDonnell et al have observed in rats that Bcl-2 mRNA increased to its maximum levels 10 days following castration. These results along with our findings in this study strongly indicate that androgen ablation therapy may increase Bcl-2 in prostate cancer. Bcl-2 overexpression may reach such a high level at some point that the balance between apoptotic rates and Bcl-2 expression is tipped in favor of resistance to apoptosis; eventually resulting in resistance to hormonal therapy in the androgen independent tumor. An alternative explanation of increased Bcl-2 expression following androgen ablation therapy is that while hormonal-ablation therapy induces cell death via apoptosis, clones of high Bcl-2 expressing apoptosis-resistant cells which exist even before the treatment may expand during the treatment and finally become the majority of the tumor. 53 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Interestingly, it has been recently shown that Bcl-2 induces angiogenesis through vascular endothelial growth factor (VEGF) in mouse models; this activity of Bcl-2 is independent of its anti-apoptotic activity [Fernandez et al. 2001], Thus, Bcl- 2 may contribute to the development of androgen independence through a combination of apoptosis resistance and angiogenesis. We have shown in the previous study (Chapter 2) that the expression of CDK-inhibitor p27 is of prognostic significance in prostate cancers; tumors expressing high levels of p27 showed an increased recurrence fiee survival and overall survival compared to those expressing low levels of p27 [Cote et al. 1998]. Our results in this study suggest that p27 expression is induced by hormone ablation therapy (Figure 3-5). Thus, the initial response to hormonal ablation therapy appears to be in part due to decreased cell proliferation rates that result from increased p27 expression. This result is supported by the study of Knudsen et al [Knudsen et al. 1998]. They have demonstrated in the hormone-sensitive cell line LNCaP, that propagation in the absence of androgen significantly increased p27 protein levels and arrested cell cycle progression at G1 phase, as compared with cells propagated in complete serum. We further show in this study that the development of androgen independence is associated with a dramatic decrease in p27 expression (Figure 3-5). Since p27 is a cell cycle inhibitor, decreased p27 expression enhances cell cycle progression through Gl/S phase that results in higher cell proliferation rates. 54 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Therefore, decreased p27 expression and increased proliferation protein Ki-67 expression observed in this study indicate that androgen independent tumor cells maintain their high proliferative ability even in an androgen-deprived environment. Decreased p27 expression may contribute to the development of androgen independence through loss of cell cycle control. Several studies have shown that the expression of p27 is high in cells inhibited by serum deprivation and declines upon mitogenic stimulation [Nourse et al. 1994; Knudsen et al. 1998]. We postulate that alternative growth pathways may turn on and decrease p27 expression that enables the prostate cancer cells to proliferate in presence of androgen deprivation. Based on these findings, we propose a model for the development of hormonal resistance in prostate cancer. Hormone naive prostate cancers show low Bcl-2, and moderate to high p27 expression. Hormonal ablation therapy results in increased apoptosis and p27 expression. These factors allow for the initial response to hormonal ablation through increased cell death and decreased cell proliferation. However, induction of Bcl-2 occurs simultaneously, and may be a harbinger of inevitable hormonal resistance. The development of hormonal resistance is a result of (i) resistance to apoptosis through increased Bcl-2 expression (with perhaps, a Bcl-2 induced increase in angiogenesis), (ii) high cell proliferative ability because of decreased p27 expression that may due to availability of alternative growth mechanisms (Figure 3-7). 55 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3-1. Representative apoptosis detected by in situ end labeling on formalin-fixed, paraffin-embedded tissue sections of the untreated and treated prostate cancers. A). Low apoptotic index detected in the untreated prostate cancers B). High apoptotic index detected in the treated prostate cancer. Note the nuclear irregularities and chromatin condensation characteristic of apoptotic cells. The majority of tumors that underwent preoperative hormonal ablation therapy demonstrate a greater apoptotic index than that seen for many of the cases not exposed to preoperative hormonal ablation therapy. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3-2. Apoptotic indices of stage C prostate cancer: Comparison of patients treated preoperatively with DES (3 days to 20 weeks) with those not exposed to preoperative hormonal treatment. The result of the apoptotic index for each patient is represented by a point. Note that the majority of the treated cases (80%) demonstrate an apoptotic index greater than the baseline median apoptotic index of 0.75. The majority of tumors that underwent preoperative hormonal ablation therapy demonstrate a greater apoptotic index than that seen for many of the cases not exposed to preoperative hormonal ablation therapy. 6 -r 5 - 1 - - 0.75 ' * 0 ’ • * * w “" o ■ ■ ! ■ ■ ■ * * ----------------- 1 — * * ° ° * .......- ...... ....... H o r m o n e - T r e a te d H o r m o n e - U n tr e a te d 58 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3-3 Representative immunoreactivity of Bcl-2, p27 and Ki-67 in prostate cancer. A) Tumor demonstrating high Bcl-2 expression (>10% of tumor cell). B) Tumor demonstrating low Bcl-2 expression (<10% of tumor cell). Note the lymphocytes indicated by arrowhead express Bcl-2 protein, used as an internal control. C) Tumor demonstrating high p27 expression (>10% of tumor cell). D) Tumor demonstrating low p27 expression (<10% of tumor cell). Note the adjacent normal prostate gland expresses p27 protein, used as an internal control. E) Tumor demonstrating high Ki-67 expression. F) Tumor demonstrating low Ki-67 expression. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 60 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 3-1. Bcl-2 expression in the untreated, treated and androgen independent (Al) group of prostate cancer. Untreated Group: tumors from thirty-one patients with pathologic stage C disease who had no exposure to androgen ablation therapy prior to radical prostatectomy. Treated Group: tumors from thirty patients with pathologic stage C disease who received androgen ablation therapy prior to radical prostatectomy. Al Group: tumors from twenty patients with advanced, hormone- resistant (androgen independent) prostate cancer who had rising PSA despite orchiectomy and systemic hormone therapy. Bcl-2 immunoreactivity: the tumors were categorized into one of the following two groups: low Bcl-2 expression, which was defined as less than or equal to 10% (0-10%) of tumor cell showing membrane immunoreactivity equal to or stronger than that of the basal cell of the adjacent normal prostate glands; and high Bcl-2 expression, which was defined as more than 10% (>10%) of tumor cell showing cytoplasmic immunoreactivity. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Bcl-2 Expression Low (%) High (%) Total (%) P value (Chi square test) Untreated Group Treated Group 20 (90) 15 (50) 2(9) 15 (50) 22 (100) 30(100) 0.002 Treated Group Al Group 15 (50) 8 (29) 15 (50) 20 (71) 30 (100) 28 (100) 0.096 Untreated Group Al Group 20 (90) 8(29) 2(9) 20 (71) 22 (100) 28 (100) 0.00001 Chi square test: p < 0.001 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3-4. Bci-2 expression in the untreated, treated and androgen independent (AI) group of prostate cancer. Untreated Group: tumors from thirty- one patients with pathologic stage C disease who had no exposure to androgen ablation therapy prior to radical prostatectomy. Treated Group: tumors from thirty patients with pathologic stage C disease who received androgen ablation therapy prior to radical prostatectomy. AI Group: tumors from twenty patients with advanced, hormone-resistant (androgen independent) prostate cancer who had rising PSA despite orchiectomy and systemic hormone therapy. High Bcl-2 immunoreaetivity: more than 10% (>10%) of tumor cell showing cytoplasmic immunoreactivity of Bcl-2. § 0.7 • tfl 0.5 - £ J Z g 0.4 - 1 S 0.3 - 1 0 % s • S 0 2 - 0 c n n S 0.1 A Q a 0 .0 - 1 U ntreated G roup T reated G roup Chi>square test, p < 0.001 AI Group 63 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 3-2. p27 expression in the untreated, treated and androgen independent (AI) group of prostate cancer. Untreated Group: tumors from thirty-one patients with pathologic stage C disease who had no exposure to androgen ablation therapy prior to radical prostatectomy. Treated Group: tumors from thirty patients with pathologic stage C disease who received androgen ablation therapy prior to radical prostatectomy. AI Group: tumors from twenty patients with advanced, hormone- resistant (androgen independent) prostate cancer who had rising PSA despite orchiectomy and systemic hormone therapy. p27 immunoreactivity: the tumors were categorized into one of the following two groups: low p27 expression, which was defined as less than or equal to 10% (<10%) of tumor cell showing membrane immunoreactivity equal to or stronger than that of the basal cell of the adjacent normal prostate glands; and high p27 expression, which was defined as more than 10% (>10%) of tumor cell showing nuclear immunoreactivity. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. P27 Expression Low (%) High (%) Total (%) P value (Chi square test) Untreated Group Treated Group 18(58) 8(28) 13 (42) 21 (72) 31 (100) 29 (100) 0.04 Treated Group AI Group 8(28) 22 (85) 21 (72) 4(15) 29 (100) 26 (100) 0.00001 Untreated Group AI Group 18(58) 22 (85) 13 (42) 4(15) 31 (100) 26 (100) 0.002 Chi square test: p < 0.001 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3-5. p27 expression in the untreated, treated and androgen independent (AI) group of prostate cancer. Untreated Group: tumors from thirty-one patients with pathologic stage C disease who had no exposure to androgen ablation therapy prior to radical prostatectomy. Treated Group: tumors from thirty patients with pathologic stage C disease who received androgen ablation therapy prior to radical prostatectomy. AI Group: tumors from twenty patients with advanced, hormone- resistant (androgen independent) prostate cancer who had rising PSA despite orchiectomy and systemic hormone therapy. High p27 immunoreactivity: more than 10% (>10%) of tumor cell showing nuclear immunoreactivity. Compared to the untreated group, p27 expression was increased in the treated group and decreased in 0.6 - 0.5 - 0.3 - * 0.1 72% \ 42% 15% 0.0 Untreated Group Treated Group Ai Group Chi square test, p < 0.001 66 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3-6. Mean level of Ki-67 expression in the untreated, treated and androgen independent (AI) group of prostate cancer. Untreated Group: tumors from thirty-one patients with pathologic stage C disease who had no exposure to androgen ablation therapy prior to radical prostatectomy. Treated Group: tumors from thirty patients with pathologic stage C disease who received androgen ablation therapy prior to radical prostatectomy. AI Group: tumors from twenty patients with advanced, hormone-resistant (androgen independent) prostate cancer who had rising PSA despite orchiectomy and systemic hormone therapy. Ki-67 immunoreactivity: average number of tumor cells showing nuclear immunoreactivity of Ki-67 counted in 10 high power fields (X400). Compared to the untreated and treated group, Ki-67 expression was increased dramatically in androgen independent group. * O J L _ r_ L U n tre a te d G r o u p T r e a te d G r o u p AI G ro u p p = 0 .0 0 4 67 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3-7. Model of coordinate molecular changes following androgen ablation therapy and androgen independence. A. Hormone nai've prostate cancer cells (Untreated group) are dependent on androgen for growth and survival. B. Hormonal ablation therapy decreases androgen available to androgen dependent prostate cancer cells and results in increased apoptosis and p27 expression. However, induction of Bcl-2 occurs simultaneously, and may be a harbinger of inevitable hormonal resistance. C. The development of hormonal resistance is a result of (i) resistance to apoptosis through increased Bcl-2 expression which may due to availability of alternative survival mechanisms (ii) high cell proliferative ability because of decreased p27 expression which may due to availability of alternative growth mechanisms. A. A n d ro g en Cell Growth Androgen Androgen Dependent Tumor (Untreated Group) A n d ro g en 68 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. B. Androgen Cell Growth Cell Survival S C 4 Androgen Androgen Dependent Tumor (Treated Group) f Bcl-2 c. Androgen * "ell Growth \ j & Alternative Growth And Survival Stimulus Bcl^l'Nlr Androgen Independent Tumor (AI Group) Cell Survival * Androgen 69 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter four: Her 2/neu expression in prostate cancer: high level of expression associated with exposure to hormone therapy and androgen independent disease 4.1 Introduction HER 2/neu (c-erb B-2) is a proto-oncogene located at chromosome 17q. [Ross et al. 1998] It encodes a transmembrane receptor with tyrosine kinase activity, which belongs to the family of epidermal growth factor (EGF) receptors. [Ross et al. 1998] Although its specific ligand has not been identified, Her 2/neu has been found important in the regulation of cell growth and differentiation. [Klapper 2000] Her 2/neu has been extensively studied in breast cancer [Ross et al. 1998], It has been demonstrated that protein overexpression (which is usually due to gene amplification), is inversely correlated with estrogen receptor level, and predicts resistance to anti-estrogen therapy, even in the case of estrogen receptor positive disease. [Adnane et al. 1989; Zeillinger et al. 1989; Newby et al. 1997; Elledge et al. 1998] Her 2/neu may provide an alternative growth pathway for breast cancer cells, especially in the case of estrogen-independent growth. Therefore, it is not surprising that breast cancers showing HER 2/neu gene amplification and/or protein overexpression have a worse clinical prognosis. [Ross et al. 1998] Based on these data from breast cancer, there has been speculation that Her 2/neu may also contribute to the development of hormone resistance in prostate 70 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cancer. Using a prostate cancer xenograft model, Craft et al [Craft et al. 1999] demonstrated that the androgen-independent xenografts of LAPC-4 showed higher Her 2/neu protein levels than the androgen dependent LAPC-4. Forced overexpression of Her 2/neu in the androgen dependent LNCaP cells induced androgen-independent growth in vitro, and accelerated progression to androgen- independence in castrated animals. [Craft et al. 1999] In contrast to breast cancer, where estrogen insensitivity is often associated with loss of expression of the estrogen receptor, [McGuire et al. 1991] the majority of androgen independent prostate cancers maintain their androgen receptor expression and androgen dependent gene expression, such as PSA. [Visakorpi 1999] This indicates that the androgen growth pathway is still active in androgen independent prostate cancer, at least in many cases. It is suggested that there may be a “cross-talk” between the Her 2/neu and androgen receptor pathway through tyrosine and MAP kinase, through which Her 2/neu can activate the androgen receptor in the absence of androgen and enhance the magnitude of the response of the androgen receptor even when exposed to low levels of androgen. [Craft et al. 1999; Yeh et al. 1999] Although Her 2/neu gene amplification and protein expression have been investigated in human prostate cancer, the results are controversial. Ross et al investigated 113 cases of prostate cancer by FISH and found 41% of them showing HER-2/neu gene amplification. [Ross et al. 1997] However, Pauletti et al was unable to demonstrate gene amplification among 125 cases of prostate cancer, including 71 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cases of advanced diseases. [Pauletti 2000] The frequency of Her 2/neu protein overexpression is reported from 0% to 100% in prostate cancers and the effect of Her 2/neu expression on the outcome is also undefined. [Mellon et al. 1992; Visakorpi et al. 1992; Zhau et al. 1992; Gu et al. 1996] This variation in results is probably due to the heterogeneity of prostate cancer, biological differences in study cohorts, and methodological differences. With this in mind, we performed the present study by involving only pathologic stage C and advanced, androgen independent prostate cancer. We designed this cohort specifically to minimize confounding factors that might influence Her 2/neu expression. In addition, patients with stage C disease have higher recurrence rates than those with stage B disease, even though they are all classified as clinical localized cancer (no lymph node and systemic metastasis). Thus, studies on stage C disease are more likely to identify the biologic differences among tumors that are important in disease recurrence. The purpose of the present study was to examine: (1) Her 2/neu expression in primary, androgen dependent and advanced, androgen independent prostate cancer; (2) the effect of androgen ablation therapy on the expression of Her 2/neu in prostate cancer; (3) the association of Her 2/neu expression with other intermediate prognostic markers, including Gleason score and substage; (4) the association of Her 2/neu expression with prostate cancer progression. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.2 Materials and Methods 4.2.1 Patient Population The study included material from eighty-one patients with prostate cancer, including sixty-one cases of pathologic stage C (pT3, NO, MO) prostate cancer [1992] who received radical retropubic prostatectomy with bilateral pelvic lymph node dissection at the USC/Norris Comprehensive Cancer Center between 1982 and 1996, and 20 cases of advanced prostate cancer who had rising prostate specific antigen (PSA) despite hormonal ablation via orchiectomy and systemic hormone therapy. These patients underwent transurethral resection for the relief of urinary obstruction at Ruhr University, Bochum, Germany between 1990 and 1992. According to their treatment status, patients were subdivided into the following groups: (1) Untreated group (46-79 years, median age = 65 years): tumors from thirty-one patients with pathologic stage C disease who had no exposure to androgen ablation therapy prior to radical prostatectomy (2) Treated group (54-77 years, median = 66 years): tumors from thirty patients with pathologic stage C disease who received neoadjuvant androgen ablation therapy (Diethylstilbestrol, lmg twice or four times per day) prior to radical prostatectomy. The duration of DES treatment varied from 3 days to 20 weeks. This group was hormone treated, but considered to be androgen responsive. (3) Androgen independent group (50 - 91 years old, median age = 74 years): tumors from twenty patients with advanced, androgen independent prostate cancer that had rising PSA despite orchiectomy and systemic anti-androgen 73 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. prostate cancer that had rising PSA despite orchiectomy and systemic anti-androgen or androgen ablation therapy (Fifteen patients had cyproterone acetate 50 - 150 mg daily. The duration varied from 2 to 96 months. Another two patients had received LHRH analogue for 2 years, 3.6 mg per month. The remaining two patients had orchiectomy but did not have any systemic hormone therapy). All tumors were graded according to the Gleason system. [Gleason 1977] Gleason scores were matched between the untreated and treated group. The substage of disease was categorized as described by Gibbons et al: [Gibbons 1986] Cl- invasion through capsule without involvement of surgical margin or seminal vesicles, C2- a positive surgical margin without seminal vesicle involvement, and C3-involvement of seminal vesicle(s). Because seminal vesicle invasion usually confers a worse outcome, we stratified the 61 cases of stage C prostate cancer into two groups: C1/C2 vs. C3. 4.2.2 Patient Follow-up All subjects were evaluated at 1, 2 and 6 months after surgery, followed by 6- month intervals until postoperative year 5, and annually thereafter. Clinical recurrence of cancer was determined by biopsy. Metastatic disease was determined by bone scan or other clinical findings. PSA recurrence was determined by finding serum PSA level equal or more than 0.4 ng/mL on two consecutive tests. The median follow-up time for the 31 cases in the untreated group was 12.7 years. 74 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.2.3 Immunohistochemistry 5-urn-thick sections from formalin-fixed, paraffin-embedded prostate cancer tissue blocks were mounted on Probe-On Plus slides (Fisher Scientific, Pittsburgh, PA). The slides were deparaffinized with xylene, rehydrated through 100% and 95% ethanol. Endogenous peroxidase was quenched in 3% hydrogen peroxide in absolute methanol. Antigen retrieval [Shi et al. 1997] was performed by soaking slides in citrate buffer (pH = 6) and heated in 90°C water bath for 25 minutes. The slides were then cooled for 20 minutes at room temperature. After being blocked for nonspecific binding with 5% normal goat serum (20 minutes), slides were incubated overnight at room temperature with anti-Her 2/neu polyclonal antibody (DAKO Corp., Carpenteria, CA, the same antibody used for the HercepTest™) at a dilution of 1:600 in phosphate-buffered saline (PBS). The antibody is produced by immunizing a rabbit with the synthetic human c-erbB-2 oncoprotein peptide from the intracytoplasmatic part of the c-erbB-2 oncoprotein (DAKO Corporation, Data sheet). PowerVision™ Two-Step Histostaining Kit (ImmunoVision Technologies, Co., Daly City, CA) was used to detect the immunoreactivity on the slides as described previously. [Shi et al. 1999] Briefly, slides were incubated with Poly-horse radish peroxidase (HRP)-Goat x Rabbit IgG for 20 minutes. After washing with PBS, the slides were incubated with diaminobenzidine (DAB) for 10 minutes. Hematoxylin was used for counterstaining. 75 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Paraffin sections from the breast cancer tissue block which were known positive for Her 2/neu were used as positive controls and they were also used as negative controls when incubated with PBS instead of anti-Her 2/neu antibody in each immunohistochemistry run. 4.2.4 Assessment of Immunoreactivity All slides were interpreted by two investigators (R.J. Cote and Y. Shi) independently who were blinded to all outcome data. The discrepant cases were re assessed until concordance was reached. Her 2/neu membrane immunoreactivity was observed in the basal cells of non-malignant prostate glands, which served as internal positive controls to monitor the quality of the immmunohistochemical reaction and to assess Her 2/neu expression (Figure 1 A) in each case. Based on the percentage of tumor cells showing Her 2/neu membrane immunoreactivity equal or stronger than that of the basal cell of the adjacent normal prostate glands, the tumors were categorized into one of the following two groups: low Her 2/neu expression, which was defined as less than or equal to 50% (<50%) of tumor cells showing membrane immunoreactivity equal or stronger than that of the basal cell of the adjacent normal prostate glands; and high Her 2/neu expression, which was defined as more than 50% (>50%) of tumor cell showing membrane immunoreactivity equal or stronger than that of the basal cell of the adjacent normal prostate glands. In all cases, tissue sections from the most representative area of tumors were selected for assessment. The level of Her 2/neu expression was based 76 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. on the overall assessment of the entire tissue section examined at 40x and lOOx power (Olympus BH-2 microscope, Melville, NY). 4.2.5 Statistical Analysis Fisher’s exact test was used to compare Her 2/neu expression in various patients groups (e. g. untreated groups vs. treated group vs. androgen independent group; low Gleason score vs. high Gleason score; low substage vs. high substage). Kaplan-Meier plot and the logrank test were performed to assess the association of Her 2/neu expression with overall survival and time to clinical and/or PSA recurrence in the untreated group. The results were considered significant if the two- sided p value was less than 0.05. 4.3 Results 4.3.1 Her 2/neu expression in normal prostate epithelium Her 2/neu membrane immunoreactivity was observed in the basal cells but not the luminal cells of normal prostate glands, which served as internal controls for the assessment of Her 2/neu expression. Her 2/neu expression was also observed in the basal and luminal cells of hyperplastic glands (Fig 1A). The finding of Her 2/neu expression in prostate basal cells and by hyperplastic prostate epithelium has been described [Schwarta 1998; Haussler et al. 1999]. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.3.2 Her 2/neu expression in localized prostate cancer Her 2/neu expression in the untreated and treated groups was investigated. Of 31 cases in the untreated group, 9 (29%) showed high Her 2/neu expression (Figure IB and C, Table 1). In contrast, of 30 cases in the treated group, 15 (50%) showed high Her 2/neu expression (Figure ID, Table 1). This difference did not quite reach statistical significance (p = 0.120). 4.3.3 Her 2/neu expression in advanced androgen independent prostate cancer Of 20 cases of the advanced, androgen independent prostate cancer, 17 (85%) showed high Her 2/neu expression (Table 1). Compared to the untreated and treated group, Her 2/neu expression was significantly elevated in androgen independent prostate cancer (p < 0.001). The difference of Her 2/neu expression was still significant when it compared separately with the untreated and treated group (p < 0.001 and p = 0.016, respectively). In addition, the intensity of Her 2/neu immunoreactivity was also increased in the androgen independent group of prostate cancer (Figure IE and F). Almost all the cases of this group showed strong Her 2/neu membrane immunoreactivity. 4.3.4 Association of Her 2/neu expression with Gleason score and tumor substage In order to assess the potential clinical value of Her 2/neu expression in prostate cancer, we analyzed the association of Her 2/neu immunoreactivity with Gleason score and substage of prostate cancer. Among the 31 cases in the untreated 78 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. group, there was a significant association between Her 2/neu expression and Gleason score (p = 0.038); tumors with high Gleason scores (Gleason score 8-10) tended to have high Her 2/neu expression (Table 2). The level of Her 2/neu expression was not found to be associated with Gleason score of the tumors in the treated group (p = 0.700, Table 3). Among the tumors with low/moderate Gleason score (5 - 7), high Her 2/neu expression was observed in 15% of the untreated group, vs. 55% of the treated group (p = 0.019, Table 2 and 3). Among the tumors with high Gleason score (8-10), high Her 2/neu expression was observed in 54% of the untreated group, vs. 40% of the treated group (Table 2 and 3). Thus, the effect of androgen ablation therapy on increased Her 2/neu expression in primary prostate cancer was most pronounced in tumors of low to moderate Gleason score. No significant association was found between Her 2/neu expression and tumor substage in both untreated and treated groups (p = 0.217 and p = 1.000, respectively, Table 2 and 3), although it is recognized that the sample size is small. 4.3.5 Association of Her 2/neu expression with recurrence and survival In order to further assess the value of Her 2/neu expression as a marker of progression in prostate cancer, we evaluated the association of Her 2/neu immunoreactivity with overall survival and recurrence risk in the untreated group. In this analysis, we only studied tumors from the untreated group, as the results indicated that exposure to androgen ablation therapy altered the Her 2/neu expression pattern (see above). Among 31 cases from the untreated group, there was a 79 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. significant association between Her 2/neu expression and overall survival (p = 0.044); patients with tumors showing high Her 2/neu expression tended to have a lower overall survival chance (Figure 4-2A). At the median follow-up year 12, the survival rate for the cases with low vs. high Her 2/neu expression was 71% vs. 44% respectively. Although the association between Her 2/neu expression with recurrence risk (clinical and/or PSA recurrence) did not reach statistical significance (p = 0.221), there was a trend that the cases with low Her 2/neu expression had lower recurrence risk (Figure 4-2B). At the median follow-up year 12, the possibility of remaining recurrence free for the cases with low level of Her 2/neu expression was 61% vs. 42% for those with high Her 2/neu expression. Again, we recognize that the sample size is small. 4.4 Discussion Despite advances in anti-cancer therapy, androgen independence is still the final result for advanced prostate cancer treated with hormonal therapy. [Sinha et al. 1977] The mechanisms responsible for the development of androgen independence are unknown. It is postulated that Her 2/neu may contribute to the development of androgen independence in prostate cancer. [Craft et al. 1999] In this study, we investigated Her 2/neu expression in three patient cohorts; never exposed to hormone therapy, exposed but sensitive to hormone therapy, and exposed but resistant to hormone therapy. These cohorts were designed to represent different stages of development of androgen independence in prostate cancer, and represent a unique 80 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. aspect of this study. We observed progressive elevation in the level of Her 2/neu expression from the androgen sensitive untreated cancer, through the androgen sensitive treated cancer to androgen independent cancer. High level of Her 2/neu expression is a frequent event in androgen independent prostate cancer, and is significantly associated with the development of androgen independence. This finding is consistent with a recently published report from Signoretti et al, [Signoretti et al. 2000] and it is also consistent with previous reports that metastatic prostate cancer demonstrate higher Her 2/neu expression compared with localized cancers, and that patients with end-stage of prostate cancer have higher serum concentration of Her 2/neu [Arai et al. 1997; Morote et al. 1999]. It is not clear how Her 2/neu contributes to the development of androgen independence; high Her 2/neu expression may provide a survival and proliferation advantage for tumor cells in an androgen depleted environment through activation of the androgen receptor in the absence of ligand, as described by Craft et al [Craft et al. 1999]. The effect of androgen ablation therapy on Her 2/neu expression has at least two possible explanations. One possibility is that androgen ablation therapy may induce (up-regulate) Her 2/neu expression in androgen sensitive prostate cancers in order to compensate for the loss of growth stimulation; this may occur through Her 2/neu tyrosine kinase activation of the androgen receptor through an intermediate activator pathway, such as the MAP kinase pathway [Craft et al. 1999; Yeh et al. 1999]. In the case of breast cancer, Antoniotti et al found that anti-estrogen therapy 81 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. with tamoxifen or estrogen deprivation can up-regulate Her 2/neu expression in estrogen-responsive breast cancer cell lines [Antoniotti et al. 1992], Alternatively, Craft et al have proposed that androgen independent clones may exist at low frequency in androgen-dependent prostate cancers [Craft et al. 1999]. Anti-androgen therapy may induce the selective outgrowth of these cells, of which one characteristic may be elevated Her 2/neu protein expression. The present study demonstrates that high Her 2/neu expression can be seen even after initial exposure to androgen ablation therapy. These observations may support the idea that up- regulation of Her 2/neu expression or clonal outgrowth of Her 2/neu expressing cells is an early event in the response of prostate cancer cells to androgen ablation therapy. Although HER-2/neu gene amplification has been observed in 25-30% of breast and ovarian cancers [Slamon et al. 1989], several studies have found this is not the case in prostate cancer, where the frequency of HER-2/neu gene amplification is much lower than in breast and ovarian cancer [Zhau et al. 1992; Mark et al. 1999; Pauletti 2000]. In prostate cancer, Her 2/neu protein expression may be regulated mainly at the transcription and post-translation level. This is also the major regulatory mechanism for p27 expression in prostate cancer [Cote et al. 1998]. Thus, for these molecular markers (p27, Her 2/neu), immunohistochemistry is an important tool to investigate their association with tumor progression and response to therapy. Increased expression of Her 2/neu appears to be a regulatory event in response to environmental change, such as androgen ablation. Increased Her 82 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2/neu expression in prostate cancer does not appear to result from genetic alteration (such as gene amplification) [Pauletti 2000]. As such, we speculate that while Her 2/neu expression may play an important role in the development of androgen resistance, it may not be an early event involved in the development of prostate cancer. We observed Her 2/neu expression in basal cells and hyperplastic prostate glands. This was not unexpected, and in fact has been previously described [Schwarta 1998; Haussler et al. 1999]. It is known that the biologic function of growth factors and receptors, such as Her 2/neu, can be different in tumor cells compared to their normal counterparts. Normal cells not only have growth stimulation pathways, but also, importantly, they contain intact negative regulatory pathways that serve as a “brake” on cell growth. It has been shown that in normal cells, Her 2/neu is important in differentiation [Klapper 2000]. In this study, we have shown that in early stage localized prostate cancer, the effect of hormonal therapy on increased expression of Her 2/neu occurred primarily in tumors with low to moderate Gleason score. It has been previously shown that the response to androgen ablation therapy is heterogeneous and appears to be inversely correlated to Gleason grade [Montironi 1998], with the greater response seen in the prostate cancers with lower Gleason score. We speculate that this may be due in part to the fact that low-grade tumors show the lowest endogenous levels of Her 2/neu expression. Tumors with high Gleason score, which show high levels of Her 2/neu 83 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. expression may be better able to survive and proliferate in the androgen depleted environment, and thus may not be as responsive to androgen ablation therapy. These results suggest that high level of Her 2/neu expression may be critical for the survival of prostate cancer cells in an androgen deprived environment. In this study we have shown that Her 2/neu expression is strongly associated with the development of androgen independence, and is also associated with Gleason score in untreated localized primary prostate cancer. Thus it is possible that Her 2/neu expression may be used to select a subgroup of patients who need more aggressive therapy targeting both androgen and Her 2/neu mediated growth pathways. This conclusion is also supported by the report from Agus et al [Agus et al. 1999]. They found that Herceptin (anti-Her 2/neu antibody) induced a significant growth inhibition in an androgen dependent human prostate cancer xenograft model that expressed high level of Her 2/neu protein, and that Herceptin can enhance the anti-tumor activity of paclitaxel in the androgen dependent and independent xenografts models. Thus, Her 2/neu expression may be useful in assessing the biologic behavior of prostate cancer, its potential response to anti-androgen therapy, and as a potential therapeutic target. In this regard, high levels of Her 2/neu expression in advanced androgen resistant prostate cancer may suggest the possibility of targeted therapy (i.e., Herceptin antibody therapy) in these patients, who no longer respond to hormone therapy for prostate cancer. Finally, Her 2/neu expression in prostate cancer may lead to insights into the mechanism of 84 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. development of androgen independence. Thus, assessment of Her 2/neu expression in prostate cancer may be useful in the management of this disease. 85 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 4-1. Her 2/neu expression in the Untreated, Treated and Androgen independent prostate cancer. Untreated: tumors from thirty-one patients with pathologic stage C disease who had no exposure to androgen ablation therapy prior to radical prostatectomy. Treated: tumors from thirty patients with pathologic stage C disease who received androgen ablation therapy prior to radical prostatectomy. AI: tumors from twenty patients with advanced, hormone-resistant (androgen independent) prostate cancer who had rising PSA despite orchiectomy and systemic hormone therapy. *Her 2/neu immunoreactivity: the tumors were categorized into one of the following two groups: low Her 2/neu expression, which was defined as less than or equal to 50% (<50%) of tumor cell showing membrane immunoreactivity equal or stronger than that of the basal cell of the adjacent normal prostate glands; and high Her 2/neu expression, which was defined as more than 50% (>50%) of tumor cell showing membrane immunoreactivity equal or stronger than that of the basal cell of the adjacent normal prostate glands. Her 2/neu immunoreactivity* Low level High level Total Untreated 22 (71%) 9 (29%) 31 (100%) Treated 15 (50%) 15 (50%) 30 (100%) AI 3 (15%) 17 (85%) 20 (100%) Fisher Exact test, p=0.0004 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 4-2. Association of Her 2/neu Expression with Gleason Score and Tumor Substage in Untreated Group. Untreated Group: tumors from thirty-one patients with pathologic stage C disease who had no exposure to androgen ablation therapy prior to radical prostatectomy. *Her 2/neu immunoreactivity: As in Table 1. Her 2/neu immunoreactivity* No. of subjects P value Low level High level All subjects 31 (100%) 22 (71%) 9 (29%) Gleason score 5-7 20(100%) 17 (85%) 3 (15%) 0.038 8-10 11 (100%) 5 (46%) 6 (54%) Substage C1/C2 20 (100%) 16 (80%) 4 (20%) 0.217 C3 11(100%) 6 (55%) 5 (45%) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 4-3. Association of Her 2/neu Expression with Gleason Score and Tumor Substage in Treated Group. Treated Group: tumors from thirty patients with pathologic stage C disease who received androgen ablation therapy prior to radical prostatectomy. *Her 2/neu immunoreactivity: As in Table 1. No. of subjects Her 2/neu immunoreactivity* P value Low level High level All subjects 30 (100%) 15 (50%) 15 (50%) Gleason score 5-7 20 (100%) 9(45%) 11 (55%) 0.700 8-10 10 (100%) 6 (60%) 4 (40%) Substage C1/C2 21 (100%) 10(48%) 11 (52%) 1.000 C3 9 (100%) 5 (56%) 4 (44%) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 4-1. Her 2/neu expression in prostate cancer (original magnification X400). A) The basal (arrow) and glandular cells (arrowhead) of prostate hyperplastic glands showing moderate Her 2/neu membrane immunoreactivity. B) Tumor of high Gleason grade (grade 5) from the untreated group showing high level of Her 2/neu membrane immunoreactivity (arrowhead). The adjacent normal prostate gland was indicated by the arrow. C) Tumor of low/intermediate Gleason grade (grade 3) from the untreated group showing low level of Her 2/neu membrane immunoreactivity (arrowhead). Note the adjacent normal prostate gland with basal cells, showing moderate Her 2/neu expression, used as an internal control (arrow). D) Tumor of low/intermediate Gleason grade (grade 3) from the treated group showing high level of Her 2/neu membrane immunoreactivity (arrowhead). The adjacent normal prostate gland was indicated by the arrow. E) Tumor of low/intermediate Gleason grade (grade 3) from the androgen independent group showing high Her 2/neu membrane immunoreactivity (arrowhead). F) Tumor of high Gleason grade (grade 5) from the androgen independent group showing high Her 2/neu membrane immunoreactivity (arrowhead). Note the cytoplasmic reactivity, often seen in cases of high Her 2/neu expression. This is not considered to be Her 2/neu immunoreactivity. 89 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 4-2. Association of Her 2/neu expression with prognosis of prostate cancer patients. (A) Probability of overall survival in 31 patients of the untreated group, base on Her 2/neu expression (high vs. low). The P value was based on the logrank test. Each tick mark represents a patient who was alive at the last follow-up visit. The patients with tumors showing high Her 2/neu expression tended to have a lower overall survival chance. (B) Probability of remaining recurrence free in 31 cases of untreated group, based on the level of Her 2/neu immunoreactivity (high vs. low). The P value was based on the logrank test. Each tick mark represents a patient who has no indications of recurrence at the last follow-up visit. There was a trend that the patients with tumors showing high Her 2/neu expression had higher risk of recurrence. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A. 1.00 low HER-2/neu (n=22) O £ 0.60 high HER-2/neu (n=9) 0.50 ■S 0.40 (0 | 0.30 CL 0.20 P~ 0.0438 0.10 0.00 10 15 0 5 Years from Prostatectomy B. o £ 1.00 82 v- 0.90 3 O 0.80 O ' U > 0.70 £ C 060 R S g 0.50 0.40 low H ER-2/neu (n=22) high H ER-2/neu (n=9) 0.30 0.20 ■Q (O .£2 O P - 0.2213 0.10 0.00 CL 10 0 5 15 Years from Prostatectomy 92 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter Five: Her 2/neu overexpression promotes androgen independent growth and survival in prostate cancer cells 5.1 Introduction Even though molecular mechanisms leading to androgen independence in prostate cancer are still unclear, it is believed that the development of androgen independent tumor may be a result of a release from cell cycle arrest as well as a progressive loss of apoptotic response induced by hormone therapy. While most of these tumors stop being responsive to androgen ablation therapy, they nevertheless continue to express androgen receptor (AR), which is shown to be transcriptionally active [Feldman et al. 2001]. Thus, the failure of response to androgen deprivation is probably through activation of AR through alternative growth signaling pathways. In our clinical study of prostate cancer (described in Chapter 3 and 4), we observed that increased Her 2/neu and Bcl-2 expression and decreased p27 expression are highly associated with the development of androgen independent phenotype. These observations suggest that Her 2/neu may modulate the balance between apoptosis and proliferation. It is our hypothesis that Her 2/neu activates AR and provides an alternative survival and growth pathway required by prostate cancer cells in androgen deprived conditions through modulation of Bcl-2 and p27 expression in these cancer cells. 93 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The potential importance of Her 2/neu in androgen independence has been documented by several recent studies [Arai et al. 1997; Craft et al. 1999; Morote et al. 1999; Yeh et al. 1999; Signoretti et al. 2000; Wen et al. 2000]. Craft et al have reported that androgen independent xenograft sublines express higher levels of Her 2/neu protein than their androgen dependent counterparts. HER-2/neu transfection in LNCaP cells decreased the growth suppression caused by serum starvation and shortened their latency for tumor formation by 50% in castrated mice. In addition, HER-2/neu transfected cells expressed higher PSA protein than mock transfected cells in presence of low concentration or complete absence of androgen [Craft et al. 1999]. Their results indicated that Her 2/neu overexpression contributed to the development of androgen independence in prostate cancer through ligand- independent activation or superactivation of AR pathway. Later, Wen et al also observed that Her 2/neu overexpression promoted androgen independent survival and growth in LNCaP cells. In addition, they found that the HER-2/neu transfected cells displayed higher levels of phosphorylated Akt which specifically bound to and activated AR through phosphorylation [Wen et al. 2000]. These results further support that Her 2/neu contributes to the development of androgen independence through the activation of AR in absence of androgen, possibly via PBK/Akt signaling pathway. It is known that PBK/Akt pathway is important in the regulation of cell proliferation. As reviewed in Chapter 1, activated PI3K generates 3 ’ -phosphorylated 94 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. phosphoinositides, principally phosphatidylinositol 3,4 biphosphate (PI3,4P) and phosphatidylinositol 3,4,5 triphosphate (PI3,4,5P). These PI3K-generated phospholipids then bind to Akt through its pleckstrin homology (PH) domain. The binding results in two changes. One is translocation of Akt from cytoplasm to plasma membrane; the other is a conformational alteration of Akt that makes its Thr-308 and Ser-473 accessible for phosphorylation. At the same time, PBK-generated phospholipids activate 3-phosphoinositide-dependent protein kinase (PDK) that phosphorylates Akt at Thr-308 and Ser-473. Activated Akt then enhances cell cycle progression by suppressing the activity of transcription factor AFX/Forkhead, which results in decreased expression of its target genes [Brunet et al. 1999; Kops et al. 1999; Tang et al. 1999]. One of them being the cell cycle inhibitor p27 [Medema et al. 2000], Graff et al have reported that overexpression of activated Akt in LNCaP induced a six-fold increase in xenograft tumor growth and a dramatic decrease in p27 expression [Graff et al. 2000]. Conversely, inhibition of PBK/Akt pathway results in G1 arrest with p27 up-regulation [Li et al. 1998; Collado et al. 2000]. These results suggest to us that activated PBK/Akt pathway may down regulate the cell cycle inhibitor p27. Since there is evidence for a link between Her 2/neu overexpression and Akt activation, we postulate that decrease p27 expression in androgen insensitive prostate cancer occurs through Her 2/neu overexpression and activation of PBK/Akt pathway, enabling the cancer cells to grow in androgen deprived environment. 95 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. It is well known that PBK/Akt pathway plays a critical role in anti-apoptosis. Activated Akt prevents apoptosis through phosphorylating several components of apoptotic machinery such as BAD, caspase 9 [Datta et al. 1999]. Recently, a study from Huang et al demonstrated that loss of PTEN induced not just higher levels of Akt phosphorylation, but also higher Bcl-2 expression in prostate cancer cells. Besides, they observed Bcl-2 expression is inversely correlated with PTEN expression in prostate cancer tissue [Huang et al. 2001]. This study indicates that activated Akt up-regulates Bcl-2 expression to protect cells from apoptosis. Since the study from Wen et al suggests that Her 2/neu may activate Akt [Wen et al. 2000], we postulated that Her 2/neu overexpression in prostate cancer may induce Bcl-2 expression through PBK/Akt pathway, which enables the cancer cells to survive in androgen deprived environment. In order to test the hypothesis suggested by our studies on clinical cohorts of prostate cancer and by prior work, which is that Her 2/neu expression can modulate proliferation and apoptosis, and allow prostate cancer cells to grow and survive in an androgen deprived environment through, at least in part, its activation of PBK/Akt pathway, we established a cell line model of Her 2/neu overexpression by transferring the HER-2/neu gene into the androgen responsive prostate cancer cell line LNCaP. Since as we know, the majority of androgen independent prostate cancer expresses androgen receptor and PSA, LNCaP cell line, which expresses both, is a relatively better model of androgen independent prostate cancer than other 96 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. prostate cancer cell lines, such as PC-3 and DU145, which lack expression of AR and PSA. The goals of developing this in vitro model system were to investigate whether Her 2/neu overexpression can in deed (1) promote androgen independent growth, by modulating cell cycle regulator p27 expression; (2) promote androgen independent survival, by modulating Bcl-2 expression; (3) and activate AR via PBK/Akt pathway in an androgen deprived condition. 5.2 Materials and Methods 5.2.1 Cell Culture Androgen-responsive human prostate cancer cell line LNCaP was maintained in R PM I1640 medium (Invitrogen, Calsbad, CA) containing penicillin (50 units/ml), streptomysin (50 units/mL), and 10% fetal calf serum (FCS) (Mediatech, Inc. Herndon, VA). Androgen-depleted medium was prepared by adding 10% dextran/charcoal-stripped serum (CSS) (Omega Scientific Inc., CA) instead of FCS in phenol free RPMI 1640 (Invitrogen, Calsbad, CA). All cultures were maintained in a humidified atmosphere of 5% CO2 at 37°C. 5.2.2 Transfection Parent plasmid pCDNA3 or pCDNA3 containing full length HER-2/neu cDNA (a kind gift from Dr. Charles Sawyers, University of California, Los Angeles, CA) was inserted into LNCaP cells using LipofectAMINE 2000 (Invitrogen, Calsbad, CA). Briefly, The day before transfection, LNCaP cells were plated at 97 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 6X105 per well in a 12 well plate. The next day, the cells were incubated with the mixture of 2 pg DNA and 200 pL diluted LipofectAMINE reagent. Twenty-four hours later, the cells were replated at the dilution of 1:10 into the culture dish. The mock transfected or HER-2/neu transfected LNCaP cells were selected and maintained in culture media supplemented with 500pg/mL G418 (Invitrogen, Calsbad, CA) 5.2.3 Immunohistochemistry of transfectants grown in chamber slides Mock transfected and HER-2/neu transfected cells were plated in 4-well chamber slides at 5 X 103 / well. The cells were fixed in acetone for 10 minutes, air dried and then washed 3 times with PBS for 5 min each. Non-specific binding was avoided by blocking with normal horse serum for 15 minutes and the cells were incubated with polyclonal anti-Her 2/neu antibody at the dilution of 1:600 (DAKO Corp., Carpenteria, California) overnight at 4°C. PowerVision™ Two-Step Histostaining Kit (ImmunoVision Technologies, Co., Daly City, CA) was used to detect the immunoreactivity on the slides as described previously. [Shi et al. 1999] Briefly, cells were incubated with Poly-horseradish peroxidase (HRP)-Goat x Rabbit IgG for 20 minutes. After washing with PBS, the cells were incubated in 0.05% diaminobenzidine (DAB) for 10 minutes. Hematoxylin was used for counterstaining. Paraffin sections from a known Her 2/neu positive breast cancer tissue block were used as positive controls. The slides from the same block incubated with PBS 98 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. instead of anti-Her 2/neu antibody were used as negative controls in each immunohistochemistry assay. Her 2/neu membrane immunoreactivity was quantified with the Line Profile of Image Analysis (hnage-Pro Plus, Media Cybernetics, Silver Spring, Maryland). Optical density analysis was carefully performed according to manufacturer’s instruction. Results of optical density were documented on figures under each band measured. Each number represents the average of three independent measurements. The optical density of Her 2/neu expression in mock transfected LNCaP cells was used as a baseline to compare with Her 2/neu expression in HER 2/neu transfected LNCaP. In addition, the optical density of Her 2/enu expression in the cell line model was compared with that of clinical cohorts of prostate cancer in order to confirm the concordance of the two study systems. 5.2.4 In situ hybridization The cultured cells were harvested and washed in 0.1 M phosphate buffer (PBS, pH 7.2), and centrifuged at 7,000 rpm for 5 minutes. Re-suspension of cell lines was carried out in 10% neutral buffer formalin (NBF) for 10 minutes, followed by washing in PBS and centrifugation. Cell pellets were re-suspended in PBS with a final concentration of 107 cells/ml, and fixed in 10% neutral buffered formalin for 24 hours at room temperature. Fixed pellets were processed routinely by dehydration in graded ethanol, with clearing in xylene, and embedding in paraffin blocks using an 99 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. automated processing and embedding equipment (Tissue Tek@ VIP, Sakura Finetek, Inc., Torrance, CA, USA). The transfected HER-2/neu gene was detected using SPOT-Light ™ HER-2 DNA probe and SPOT-Light ™ CISH detection kit (Zymed Laboratories Inc. San Francisco, CA). Five-pm-thick sections from formalin-fixed, paraffin-embedded prostate cancer cell line blocks prepared as described above were mounted on Probe- On Plus slides (Fisher Scientific, Pittsburgh, PA). The slides were deparaffinized with xylene for 10 minutes at room temperature, washed three times with 100% ethanol for 3 minutes each, and air dried for 10 minutes. The slides were heated in SPOT-Light ™ tissue heat pretreatment buffer for 15 minutes at 92°C, and cooled for 20 minutes at room temperature. After washing twice in PBS for 3 minutes each, the slides were covered with 100 pi SPOT-Light ™ tissue pretreatment enzyme for 5 minutes at 37°C, followed by two washes in PBS for 3 minutes each. The slides were dehydrated through 70%, 85%, 95% and 100% ethanol. After drying in the air, the cell nuclei were hybridized with Digoxigenin labeled HER-2/neu probe (SPOT-Light ™ HER-2 DNA probe) in a humidified box overnight at 37°C. The next day, the slides were washed in 0.5 X Saline-sodium citrate (SSC) for 5 minutes at 78°C and then washed in PBS with 0.025% Tween-20 (PBST) for 3 minutes X 3. Endogenous peroxidase activity was quenched by incubation the slides in 3% hydrogen peroxide for 10 minutes. After blocking nonspecific binding with SPOT-Light ™ blocking solution for 10 minutes, the slides were incubated at room temperature first in SPOT- 100 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Light ™ FITC-sheep anti-digoxigenin antibody and then in SPOT-Light ™ HRP- goat anti-FITC antibody for 1 hour each. After washing with PBS, the slides were incubated with 0.05% diaminobenzidine (DAB) for 10 minutes. Hematoxylin was used for counterstaining. As for immunohistochemistry, known Her 2/neu positive breast cancer tissue sections were used as positive controls. 5.2.5 Cell growth analysis Mock transfected and HER 2/neu transfected LNCaP cells were seeded in T25 flask at 3 X 105 cells/flask. The medium was replaced after 48 hours by phenol free RPMI 1640 containing either 10% CSS or 10% FCS. Following this, the medium was changed every other day. The total number of mock and HER 2/neu transfected LNCaP cells in each flask were counted every day with Neubauer’s chamber (Buffalo, NY). The growth curves were made according to the average of three independent experiments. 5.2.6 Flow cytometry Mock and HER-2 transfected cells were seeded in T25 flask at 3 X 105 /flask. The medium was replaced after 48 hours by phenol free RPMI 1640 containing either 10% CSS or 10% FCS. After growing the cells with change of medium every other day in CSS for 6 days or in FCS for 3 days, they were harvested by trypsinization and suspend in 5 ml PBS. The cells were then fixed in freshly prepared ice-cold 70% ethanol for 3 hours, pelleted by centrifugation and re- 101 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. suspended in 1 ml propidium iodide (PI)/ Triton X-100 solution with RNase A (20 pg PI, 1 pL Triton X-100 and 200 pg RNAse A in 1 ml PBS). After staining for 30 minutes at room temperature flow cytometry was performed using a FACStar Plus flow cytometer (Becton Dickinson, Franklin lakes, NJ). Data were analyzed using Cellquest software (Becton Dickinson, Franklin lakes, NJ). 5.2.7 Western blot analysis Mock transfected and HER-2 transfected cells were seeded in T25 flask at the density of 3 X 105 /flask. The medium was replaced after 48 hours by phenol free RPMI 1640 containing either 10% CSS or 10% FCS. After growing the cells with change of medium every other day in CSS for 6 days or FCS for 3 days, the cells were harvested, washed with PBS and lysed in lml ice-cold RIP A buffer (150 m M NaCl, 1.0% NP-40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate, 50 mM Tris) containing protease inhibitors (35 pg/ml PMSF, 0.3 mg/ml EDTA, 0.7 pg/ml Pepstatin A, 0.5 pg/ml Leupeptin). The amount of protein in each sample was determined using Micro BCA ™ protein assay reagent kit (Pierce, Rockford, IL). Same amount (20-50 pg) of total protein from each sample was electrophoresed in SDS-PAGE gels (4-15% gradient Tris-HCl gel, Bio-Rad Laboratories, Hercules, CA). Following electrophoresis, the protein bands were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories, Hercules, CA). The membranes were blocked with 5% nonfat dry milk in Tris buffered solution containing 0.05% Tween 20 (TBST) for 1 hour at room temperature, and then 102 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. incubated with monoclonal anti-Her 2/neu antibody (BioGenex, San Ramon, CA) at the dilution of 1:400, or polyclonal anti-phosphorylated Akt antibody (Ser 473) (Cell Signaling, Beverly, MA) at the dilution of 1:500; or polyclonal anti-p27 antibody (c- 19, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at the dilution of 1:500, or polyclonal anti-PSA antibody (DAKO Corp., Carpenteria, California) at the dilution of 1:5000; or monoclonal anti-Bel 2 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at the dilution of 1:200, or monoclonal anti-|3-actin antibody (Sigma, St. Louis, MO) at the dilution of 1:5000. The incubation with each antibody listed above was carried out overnight at 4 °C. After three washes with TBST for 5 minutes each, the membranes were incubated with HRP-conjugated goat anti-mouse antibody (Bio- Rad Laboratories, Hercules, CA) at 1:3000 or goat anti-rabbit (Bio-Rad Laboratories, Hercules, CA) at 1:5000 for 2 hours at room temperature. After 3 washes with TBST 5 minutes each, the membranes were incubated with ECL substrate (Pierce, Rockford, IL) for 5 minutes. The chemiluminescent signals were detected by exposure to blue-light sensitive film (Pierce, Rockford, IL). All Western blots were quantified for protein expression with the Line Profile of Image Analysis (Image-Pro Plus, Media Cybernetics, Silver Spring, Maryland). Optical density analysis was performed according to manufacturer’s instructions. Numbers below every band in the electrophoregrams represented the average of three independent measurements. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5.2.8 in situ apoptosis detection (TUNEL assay) Mock transfected and HER-2 transfected cells were plated in 4-well chamber slides at the density of 5 X 103 / well. After 48 hours, the medium was changed to phenol free RPMI 1640 containing either 10% CSS or 10% FCS. After growth in CSS for 6 days, or FCS for 3 days, the cells were fixed with 1% paraformaldehyde in PBS for 10 minutes at room temperature. Apoptosis in each well was detected using in situ Apoptosis Detection Kit (Intergene, Purchase, NY). After two washes in PBS 5 min each, the cells were post-fixed in ethanol-acetic acid solution (ethanol : acetic acid = 2 : 1) for 5 minutes at -20°C. After washing twice with PBS 5 minutes each, the slides were incubated with 3% hydrogen peroxide for 5 minutes and washed with PBS. Subsequently, the slides were incubated with Equilibration Buffer (Intergene, Purchase, NY) for 10 seconds and then with TdT Enzyme diluted in Reaction Buffer (Intergene, Purchase, NY) for 1 hour at 37°C. After washed with the Stopping Buffer (Intergene, Purchase, NY), the slides were incubated with Peroxidase Conjugated Anti-digoxigenin antibody (Intergene, Purchase, NY) for 30 minutes at room temperature. After washing with PBS, the slides were incubated with 0.05% DAB for 5 minutes at room temperature and counterstained with hematoxylin. 5.2.9 Analysis of effects of dihydroxytestostrone (DHT) Mock and HER-2 transfected cells were seeded in T 25 flasks at 3 X 105 /flask. After 48 hours, the medium was changed to phenol free RPMI 1640 containing 10% CSS (Omega Scientific Inc., CA). The medium was changed every 104 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. other day. After the cells were grown in charcoal stripped serum for 6 days, 10 nM 5 a-dihydrotestosterone (DHT), a kind gift from Dr. Stallcup (USC Keck School of Medicine, CA) was added to the culture medium. The cells were harvested at the following time points: 3 hour, 6 hour, 15 hour, 24 hour and 48 hour. Western blot was performed to examine the effects of DHT on p27, PSA and Her 2/neu expression. The negative control cells were grown in culture medium containing the same amount of solvent, absolute ethanol, instead of DHT. 5.2.10 Statistical Analysis Paired Student’s t test was used to examine the difference of proliferation and apoptotic rates between the mock transfected and HER-2/neu transfected LNCaP cells. Chi square test was performed to examine the difference of cell cycle profiles between the mock transfected and HER-2/neu transfected LNCaP cells. The results were considered significant if the two-sided p values were less than 0.05. 5.3 Results 5.3.1 Her 2/neu protein expression in transfected LNCaP cells In order to examine the effect of transfection on Her 2/neu expression in LNCaP cells, Western blot and immunohistochemical analyses were performed. Compared to parental and the mock transfected LNCaP (LN-neo), two clones of HER-2/neu transfected LNCaP (LN-HER) cells were found to show higher HER- 2/neu expression. (Figure 5-1 A, B) Using image analysis, HER-2/neu expression 105 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. examined by Western blot in clone 1 (LN-HER Cl) was determined to be about 5 fold and that in clone 2 (LN-HER C2) 7 fold higher than the parental and mock transfected cells. The following experiments were based on the comparison of LN- HER C2 with LN-neo cells. 5.3.2 HER-2/neu gene in the transfected LNCaP In order to confirm that the increased HER-2/neu expression in the transfected LNCaP cells was due to increased HER-2/neu gene copies through stable transfection, we employed HER-2/neu gene chromogenic in situ hybridization (CISH) assay. Compared to LN-neo cells, which did not show obvious brown granules in the nuclei, LN-HER cells were found to contain more copies of HER- 2/neu gene, as evidenced by many brown granules in the nuclei (Figure 5-1 C). The result suggests that we have established a genetically modified cell clone with Her 2/neu overexpression. Since Her 2/neu gene amplification is a rare event in prostate cancer, we decided to investigate the consistency of Her 2/neu expression between the transfected cell line model and the clinical prostate cancer. The optical density of Her 2/neu expression in the untreated and androgen independent prostate cancer tissues from our clinical cohorts was 0 vs. 9.15, respectively. Similarly, the optical density of Her 2/neu expression in LN-neo and LN-HER was 0 vs. 8.25, respectively (Table 5-1). Thus, it appears that our transfected cell line model parallels the clinical 106 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. situation and can be used to study the effect of Her 2/enu overexpression on the development of androgen independence. 5.3.3 Proliferation rates in transfected LNCaP cells Since Her 2/neu overexpression has been found important in promoting cell proliferation, especially in androgen deprived conditions, we examined the proliferation rates of transfected LNCaP cells by counting the cell numbers in presence of FBS or CSS. We observed that grown in the medium containing FCS, LN-neo and LN-HER cells displayed similar proliferation rates (Figure 5-2A). The doubling time for both LN-neo and LN-HER was 48 hours. But when they were grown in the medium containing CSS, LN-neo cells show a dramatic decrease in proliferation. The doubling time for LN-neo was 96 hours or even longer after starving in CSS for more than 3 days. In contrast, LN-HER cells show a mild to moderate decrease in proliferation (Figure 5-2B). The doubling time for LN-HER cells was 72 hours. On day 6, the cell number was 13 X 105 for LN-neo cells vs. 20 X 105 for LN-HER cells. The proliferative difference in presence of CSS between these two cell clones was statistically significant (p - 0.03). This result indicates that androgen deprivation due to charcoal stripped serum induces substantial growth suppression in LN-neo cells, while Her 2/neu overexpression partially protects the cells from growth suppression caused by androgen deprivation. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5.3.4 Analysis of cell cycle profiles in transfected LNCaP cells In order to further examine the role of Her 2/neu overexpression in proliferative ability of LNCaP cells during androgen deprivation, we measured the DNA contents of the transfected LNCaP by flow cytometry (FCM). FCM showed that in presence of FCS, both LN-neo and LN-HER cells displayed similar cell cycle profiles (Figure 5-3A). The distribution of cell population for LN-neo was 58% in G l, 10% in G2 and 22% in S phase; while that for LN-HER was 61% in G l, 11% in G2 and 23% in S phase. After maintaining the cells in medium containing CSS for six days, the distribution of cell population for LN-neo was 4% in G2/M and 5% in S phase. In contrast, the distribution for LN-HER was 7% in G2/M phase and 14% in S phase (Figure 5-3B). The difference of cell population in G2/M and S phase between LN-neo and LN-HER in CSS was statistically significant (p = 0.02). This result indicates that androgen deprivation with charcoal stripped serum induces a substantial Gl arrest in the LN-neo cells, while Her 2/neu overexpression partially protects the cells from the Gl arrest caused by androgen deprivation. 5.3.5 p27 expression in transfected LNCaP cells Since p27 is a cell cycle inhibitor working at Gl/S transition, and since its expression was found to be increased by androgen deprivation in LNCaP cells, we further examined the p27 expression in tranfected LNCaP cells in presence of FCS or CSS by Western blot analysis. It was observed that both LN-neo and LN-HER cells displayed similar p27 expression in presence of FCS (Figure 5-4). After growing the 108 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cells in presence of CSS for six days, p27 expression increased 17% in LN-neo, but not in LN-HER cells. The optical density of p27 expression for LN-neo in CSS was 149.46 versus 130.69 for LN-HER. When 10 nM DHT was added back into the medium for the cells starved in CSS for 6 days (CSS 6D), p27 expression decreased in both cell lines starting at 48 hours (Figure 5-4B). The optical density of p27 expression for LN-neo decreased from 12.24 at CSS 6D to 10.01 at DHT 48 hours. The similar change was observed in LN-HER cells, where the optical density of p27 expression decreased from 11.34 at CSS 6D to 9.94 at DHT 48 hours. Since ethanol was the solvent of DHT, the cells cultured in the same volume of ethanol instead of DHT were used as negative controls. As expected, for both LN-neo and LN-HER cells, the optical density of p27 expression showed no decrease after adding ethanol in culture medium. The decrease of p27 expression was thus induced by DHT added to the culture medium. 5.3.6 Apoptotic rates in transfected LNCaP cells Since Her 2/neu overexpression has also been found to be important in promoting cell survival, especially in androgen deprived conditions, we examined the apoptotic rate in transfected LNCaP cells under both FCS and CSS conditions by TUNEL assay. The apoptotic rate was defined as mean of TUNEL positive events in 10 high power fields (hpf). We observed that in medium containing FCS, LN-neo and HER 2/neu transfected cells display similar apoptotic rate, 0.8/hpf for LN-neo cells and 0.7/hpf for LN-HER (Figure 5-5 A and B). After growth in medium 109 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. containing only CSS for 6 days, the apoptosis was increased dramatically in LN-neo, but not in LN-HER cells. The apoptotic rate was 6.2/hpf for LN-neo vs. 0.4/hpf for LN-HER cells. The difference of apoptotic rates between LN-neo and LN-HER was statistically significant (p = 0.02). This result indicates that androgen deprivation with charcoal stripped serum induces apoptosis in LN-neo cells, while Her 2/neu overexpression protects the HER 2/neu transfected cells from apoptosis caused by androgen deprivation. 5.3.7 Bcl-2 expression in transfected LNCaP Since Bcl-2 is an important apoptotic inhibitor, we examined the Bcl-2 expression in HER 2/neu tranfected LNCaP cells under both FCS and CSS conditions by Western blot analysis. LN-HER cells showed higher Bcl-2 expression than LN-neo cells in both conditions. While the optical densities of Bcl-2 expression in LN-HER were 7.16 in FCS and 6.91 in CSS; the optical densities in LN-neo were 0.04 in FCS and 0.41 in CSS. Bcl-2 expression in HER 2/neu transfected cells was about 4.6 fold higher than that in the mock transfected cells (Figure 5-6 A). This result suggests that Her 2/neu overexpression may promote androgen independent survival by upregulation of Bcl-2 expression. 5.3.8 Phosphorylated Akt in transfected LNCaP Since the study from Wen et al demonstrated that Her 2/neu overexpression activated PI in LNCaP [Wen et al. 2000], we examined the level of phospho-Akt expression in tranfected LNCaP cells under both FCS and CSS conditions by 110 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Western blot analysis. HER 2/neu transfected cells showed higher phospho-Akt expression than LN-neo cells in both conditions. Compared to LN-neo, the phospho- Akt expression in LN-HER was about 17 fold higher (Figure 5-6 B); indicating that Her 2/neu overexpression may activate Akt signaling pathway to promote androgen independent survival and growth of the transfected LNCaP cells. 5.3.9 PSA expression in transfected LNCaP Several studies have demonstrated that Her 2/neu overexpression may activate AR pathway in the absence of androgen [Craft et al. 1999; Yeh et al. 1999; Wen et al. 2000]. PSA is an androgen-regulated gene and its expression can be used as an evidence of activated AR pathway. Therefore, we examined PSA expression in tranfected LNCaP cells by Western blot analysis in presence of FCS or CSS. In presence of FCS, PSA expression was high in both LN-neo and LN-HER cells, but it was 50% higher in LN-HER cells (Figure 5-7 A). After starving the cells in CSS for 6 days, PSA expression was undetectable in LN-neo cells, while LN-HER cells continued to express PSA protein (Figure 5-7 A). This result suggests that Her 2/neu overexpression augments PSA expression in presence of androgen. In addition, it activates PSA gene despite the absence of androgen. When 10 nM DHT was added back into the culture medium for the cells starved in CSS for 6 days (CSS 6D), PSA expression was observed to increase in both cell lines. The obvious rise in PSA expression occurred at 15 hour of DHT for LN-HER, while that occurred at 24 hour of DHT for LN-neo (Figure 5-7 B). Once again, this result suggests that Her 2/neu 111 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. overexpression promotes PSA gene expression. Since ethanol was the solvent of DHT, the cells cultured in the same volume of ethanol instead of DHT were used as negative controls. As expected, for both LN-neo and LN-HER cells, PSA expression showed no increase after adding ethanol in the culture medium. This result demonstrated that the increase of PSA expression was induced by DHT that was added in the culture medium. 5.3.10 Hormone starvation increases Her 2/neu expression Since our previous study indicated that androgen ablation therapy may up- regulate Her 2/neu expression, since we constantly observed that Her 2/neu expression was increased in the cells cultured in CSS for 6 days compared to that in the cells cultured in FCS (Figure 5-4 A and B), we investigated whether the level of Her 2/neu expression can be modulated by androgen. Using Her 2/neu expression in FCS as a baseline, we found that Her 2/neu expression in LNCaP was increased about 35% when the cells were starved in CSS for 6 days (Figure 5-8 A). The optical density of Her 2/neu expression was 7.61 at FCS versus 10.90 at CSS 6D. When we added 10 nM DHT back into the medium of the cells after 6 days, Her 2/neu expression decreased from 10.90 at CSS 6D to 7.73 at DHT 48 hours (Figure 5-8 A). The similar result was observed using Her 2/neu transfected LNCaP (data not shown). Since ethanol was the solvent of DHT, the cells cultured in the medium containing equivalent volume of ethanol instead of DHT were used as negative controls. As expected, Her 2/neu expression showed no decrease after adding ethanol 112 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. in the culture medium (Figure 5-8 B). The decrease of Her 2/neu expression was thus induced by DHT in the culture medium. 5.4 Discussion It is believed that the development of androgen independence is a multi-step process. A series molecular alterations leads to alternative growth and survival mechanisms for prostate cancer cells in androgen deprived conditions induced by hormonal therapy. Her 2/neu has been extensively studied in breast cancer. It has been demonstrated that Her 2/neu overexpression contributes to the development of resistance to hormonal therapy in breast cancer [Newby et al. 1997; Elledge et al. 1998]. It is postulated that Her 2/neu may provide an estrogen independent growth pathway for breast cancer cells. Even though unlike breast cancer, where estrogen insensitivity is often associated with loss of expression of estrogen receptor [McGuire et al. 1991], majority of androgen independent prostate cancers maintain the androgen receptor expression and androgen dependent gene expression, such as that of PSA [Visakorpi 1999]. Yet, since both these two cancers are hormone- dependent, it is speculated that as in the case of breast cancer, Her 2/neu overexpression may contribute to androgen independent survival and proliferation in prostate cancer as well. Our previous studies using the clinical model comprising three cohorts of prostate cancer patients (hormone naive, hormone exposed and androgen 113 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Our previous studies using the clinical model comprising three cohorts of prostate cancer patients (hormone naive, hormone exposed and androgen independent cases) have shown that androgen independence is highly associated with increased levels of Her 2/neu expression [Shi et al. 2001]. Further, several studies from other groups have shown that Her 2/neu overexpression in prostate cancer cells may induce androgen independent survival and proliferation [Craft et al. 1999; Yeh et al. 1999; Wen et al. 2000]. More importantly, these studies suggested that Her 2/neu may activate androgen receptor in the absence of androgen through Akt or MAPK signal transduction pathways [Yeh et al. 1999; Wen et al. 2000]. These previous findings indicated that Her 2/neu plays a critical role in the development of androgen independence. In order to further analyze the Her 2/neu mediated pathway leading to androgen independence in prostate cancer, we established a LNCaP cell model of Her 2/neu overexpression by stable transfection. Like majority of androgen-independent prostate cancer as shown in our clinical cohorts, the HER- 2/neu transfected LNCaP cells retain the expression of AR and PSA, and demonstrate high levels of Her 2/neu expression. In this study, we show that compared to mock-transfected cells, HER-2/neu transfected LNCaP cells display higher proliferative rate and less G1 arrest in androgen-deprived conditions. This is consistent with several previous reports [Craft et al. 1999; Yeh et al. 1999; Wen et al. 2000]. Further, we show that HER-2/neu transfected LNCaP cells express lower p27 expression than the LN-neo cells under 114 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. androgen deprivation. Since p27 is a cell cycle inhibitor working at Gl/S transition, our results suggest that Her 2/neu overexpression protects the cells from growth suppression and G1 arrest induced by androgen deprivation probably through down- regulation of p27 expression. It has been reported that Her 2/neu antagonist such as anti-HER-2 antibody, which inhibits Her 2/neu kinase activity, increases p27 expression in breast cancer cells [Sliwkowski et al. 1999; Busse et al. 2000]. Therefore, p27 may be a critical element in Her 2/neu growth pathway connecting Her 2/neu mediated proliferative signals with cell cycle progression. The expression of p27 is regulated at both transcriptional and post- translational levels, though mainly at post-translational level [Pagano et al. 1995; Hengst et al. 1996]. There are several lines of evidence suggesting that activated Akt may down-regulate p27 expression at both these levels. Akt regulates p27 expression at transcriptional level by inhibiting its upstream transcription factor AFX/Forkhead [Medema et al. 2000]. At the post-translational level, Akt phosphorylates p27 and promotes its transport from nucleus to cytoplasm for degradation [Fujita et al. 2002]. Decreased p27 expression releases cell cycle inhibition and may induce uncontrolled cell proliferation. Therefore, we postulate that one of the molecular mechanisms of Her 2/neu overexpression leading to androgen independence is p27 down-regulation mediated by activated Akt. The decrease in p27 expression in turn, promotes cell growth in androgen deprived conditions. 115 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Data presented in this study also shows that HER-2/neu transfected LNCaP display lower apoptotic rate on hormone starvation. This observation is consistent with several previous reports [Craft et al. 1999; Yeh et al. 1999; Wen et al. 2000]. Apoptosis is a complicated cell process regulated by a balance between molecules with opposing roles such as Bcl-2, Bax. Apoptosis induction has been used as a critical therapeutic mechanism in a variety of anti-cancer treatments, including hormone therapy. Dysregulation of apoptotic pathways may therefore induce resistance to those treatments [Fisher 1994], Overexpression of anti-apoptotic proteins such as Bcl-2 has been shown to result in resistance to chemotherapy in vivo as well as in vitro [Strasser et al. 1994; Sartorius et al. 2002]. Several clinical studies including ours have demonstrated that high Bcl-2 expression is significantly associated with androgen independence and resistance to hormone therapy [McDonnell et al. 1992; Colombel et al. 1993]. In this study, we found that HER- 2/neu transfected cells demonstrated higher Bcl-2 expression than the LN-neo cells. These results suggest that, through the up-regulation of Bcl-2 expression, Her 2/neu overexpression protects the cells from apoptosis induced by androgen deprivation. This is consistent with the study of Kumar et al [Kumar et al. 1996] who observed that high Her 2/neu expression correlates with high Bcl-2 expression in estrogen receptor (ER) positive breast cancer cell lines. Her 2/neu overexpression due to stable transfection has also been show to lead to increased Bcl-2 expression and 116 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. reduced apoptosis induced by tamoxifen in ER positive breast cancer cell line MCF- 7 [Kumar et al. 1996]. Since PI3K/Akt pathways is critical survival pathway in prostate cancer cells [Lin et al. 1999; Feldman et al. 2001], the results from the present study indicate that activated Akt induced by Her 2/neu overexpression may up-regulate Bcl-2 expression, which promotes the survival of prostate cancer cell in androgen deprived conditions. In this study, we show that compared to the mock transfected cells, HER 2/neu transfected LNCaP cells display higher PSA expression in presence and absence of androgen. These results suggest that Her 2/neu overexpression induces ligand- independent AR activation. Several previous reports have documented the similar findings [Craft et al. 1999; Yeh et al. 1999; Wen et al. 2000]. Even though it is not clear how Her 2/neu activates AR, it has been shown that it may occur through MAPK and/or Akt signaling pathways [Yeh et al. 1999; Wen et al. 2000], Wen et al find that activated Akt specifically binds to AR and phosphorylated it at both Ser- 213 and Ser-791 [Wen et al. 2000]. In this study, we observe that HER 2/neu transfected LNCaP cells display higher expression of phosphorylated Akt compared to the mock transfected cells. This result suggests that PK3K/Akt pathway may be one of the mechanisms, through which Her 2/neu activates AR in absence of ligand. PTEN (phosphatase and tensin homologue deleted in chromosome 10) is a critical inhibitor of PI3K/Akt pathway. It is a lipid phosphatase working against PI3 117 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. kinase. It dephosphorylates Ptdlns (3,4,5)P3 [Wu et al. 1998], through which it inhibits Akt activation. Loss of PTEN expression occurs frequently in advanced prostate cancer [Whang et al. 1998]. Therefore, activated Akt may be a key mediator of prostate cancer progression to androgen independence and PI3K/Akt pathway could be a prime therapeutic target for patients with androgen independent prostate cancer. In the cell line model in this study, Her 2/neu expression is seen to be regulated by androgen. Her 2/neu expression in LNCaP is increased after androgen deprivation and decreased by adding DHT back into the culture media for 24 hours. These results suggest that Her 2/neu expression is down regulated by androgens in prostate cancer cells. This finding is consistent with several clinical studies including ours that increased levels of Her 2/neu expression are associated with androgen ablation therapy and the androgen independent prostate cancer developed after long term hormonal therapy displays the highest Her 2/neu expression [Signoretti et al. 2000; Shi et al. 2001]. This result is also consistent with the report from Myers et al [Myers et al. 1996]. They observed that DHT treatment at concentrations of 10"9 M or higher caused a decrease in Her 2/neu expression at both inRNA and protein levels. Their results indicate that androgens may down regulate Her 2/neu expression through direct or indirect inhibition of HER 2/neu gene transcription, or decrease the stability of HER-2/neu mRNA. Comparable down-regulation of HER-2/neu expression has also been demonstrated in several ER positive breast cancer cell lines 118 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. including T47D, MCF-7 and ZR-75-1 following estradiol treatment [Read et al. 1990; De Bortoli et al. 1992; Russell et al. 1992]. These studies also demonstrate that the regulation of Her 2/neu expression needs to go through estradiol activated ER in breast cancer cell lines [De Bortoli et al. 1992; Russell et al. 1992], While at present it is unclear that how androgen regulates Her 2/neu expression in prostate cancer, this regulation may occur via AR. Based upon our observations here, we conclude that androgen ablation therapy may induce Her 2/neu expression in prostate cancer cells, even though hormone therapy may also induce selective clonal growth of cells with high Her 2/neu expression.. Increased Her 2/neu expression in prostate cancer cells may compensate for the loss of growth and survival stimulation in androgen deprived conditions. This is comparable to findings in breast cancer, where Antoniotti et al found that anti-estrogen therapy with tamoxifen or estrogen deprivation can up- regulate HER-2/neu expression in estrogen-responsive breast cancer cell lines [Antoniotti et al. 1992]. Interestingly, it seems that not only its expression, but also the tyrosine kinase activity of Her 2/neu protein may be regulated by androgen. Meng et al have reported that prostatic acid phosphatase (PAcP), one of downstream genes controlled by androgen receptor, can dephosphorylate Her 2/neu protein on tyrosine residues in prostate cancer cells, which results in decreased proliferation rates of these cells [Meng et al. 1998]. 119 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. All these data, including ours clearly demonstrates that the “cross-talk” between AR and Her 2/neu pathways is bi-directional. Her 2/neu activates or sensitizes AR, probably through PI3K/Akt signaling pathway, especially in androgen deprived conditions. Conversely, AR activated by androgen suppresses Her 2/neu expression in presence of androgen. Part of the reason why we observed comparable growth rates and p27 expression in both mock and HER-2/neu transfected LNCaP in presence of FCS could be inhibition of Her 2/neu expression seen to occur at growth stimulating concentration of DHT. Further studies are needed to elucidate the molecular mechanisms of interaction between AR and Her 2/neu pathways. Based on our observations in this study, we propose a model of molecular mechanisms of androgen independence in prostate cancer (Figure 5-9). Her 2/neu overexpression activates alternative signal transduction pathway such as PBK/Akt pathway, through which Her 2/neu overexpression provides an alternative growth and survival stimulus for prostate cancer in androgen deprived conditions by: (1) promoting androgen independent proliferation mediated by p27 down-regulation, (2) promoting androgen independent survival mediated by Bcl-2 up-regulation; (3) activation of AR in the absence of androgen. The clinically relevant therapeutic implications of this data is that multiple points along the Her 2/neu mediated androgen independent pathway (Figure 5-9), such as Her 2/neu, PI3K and Akt, could be targeted for effecting growth inhibition of androgen independent prostate cancer cells. 120 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 5-1: Image analysis data showing similarity of Her 2/enu protein expression in clinical samples compared to HER 2/neu transfected LNCaP cell line model. Her 2/neu membrane immunostaining of the clinical samples and the cell line models were quantified using the line Profile of Image analysis (Image-Pro Plus, Media Cybernetics, silver spring, Maryland). Each numerical value of optical density represents an average of three different measurements. The optical density of Her 2/neu membrane expression for androgen independent prostate cancer was higher compared to that for the hormone naive cases. Further, the level of Her 2/neu expression in the LN-neo cells is comparable to Her 2/neu expression in the hormone naive cases, while the levels of Her 2/neu expression in high LN-HER cells are comparable to elevated Her 2/neu expression in the androgen independent cases. Clinical Cohorts of Prostate Cancer Transfected LNCaP Cell Line Model Hormone naive Androgen independent Mock transfected cells (LN-neo) HER 2/neu transfected cells (LN-HER) 0 9.15 0 8.25 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 5-1 HER-2/neu status after stable transfection with plasmid containing HER-2/neu cDNA in LNCaP. A. Western blot of Her 2/neu expression (top panel) and P-actin (bottom panel, used as a loading control for amount of total protein loaded in each lane) in the parental LNCaP, LN-neo and two clones ofHER-2/neu transfected cells. The optical densities of Her 2/neu expression in parental LNCaP, LN-neo, LN-HER Cl and LN-HER C2 were 0, 0, 5.64 , 7.37 respectively. Her 2/neu transfected LNCaP showed higher levels of Her 2/neu expression. B. Immunohistochemistry of HER-2/neu expression in LN-neo and two clones of HER-2/neu transfected cells. The cell membrane immunoreactivity was regarded as positive for Her 2/neu antibody. Her 2/neu transfected LNCaP showed higher levels of Her 2/neu expression. C. Chromogenic In situ hybridization (CISH) of HER-2/neu in the LN-neo and LN-HER cells. The brown granules in the nuclei denote positive signals. LN- neo was negative for HER 2/neu CISH staining. In contrast, brown granules were observed in the nuclei of LN-HER. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. a a t * f x I t ll 3 3 t i 6.72 f.3» M 2 6.TJ LN-aeo LK-HSR cl US-HBR c2 LN-neo L N -H E R 1 23 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 5-2 The growth curves of stably transfected LNCaP cells in the culture media containing FCS (A) or CSS (B). LN-neo: LNCaP transfected with pcDNA 3 vector. LN-HER: LNCaP transfected with recombinant HER-2/neu plasmid. (A and B) Cells were seeded at 3 X 105 in a T 25 flask. The cells were counted every 24 hours by Neubauer’s chamber. Each count represents an average of three independent experiments. In presence of FCS, LN-neo and LN-HER displayed similar proliferative rates. The doubling time was 48 hours for both of them. In CSS, the doubling time of LN-neo was 96 hours or longer versus 72 hours for LN-HER. A FCS B. CSS 35 30 —® — LN-HER o LN-neo Col 7 25 - X 20 - k. o ja £ 3 C c 8 10 - O " o O P = 0.03 2 3 4 5 6 7 0 1 0 1 2 3 4 5 6 7 Days Days 124 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 5-3. Summary of cell cycle profiles based on DNA histogram of mock transfected (LN-neo) and HER2/neu transfected (LN-HER) cells in the culture medium containing FCS (A) or CSS (B ). LN-neo: LNCaP transfected with parent vector. LN-HER: LNCaP transfected with recombinant HER-2/neu plasmid. FCS: cells were grown in RPMI 1640 containing 10% fetal calf serum. CSS6D: cells were grown in RPMI 1640 containing 10% charcoal stripped serum for 6 days. In presence of FCS, LN-neo and LN-HER displayed similar cell cycle profiles. The cell population distribution in each cell cycle phase for LN-neo was 58% in G l, 10% in G2 and 22% in S phase; while that for LN-HER was 61% in G l, 11% in G2 and 23% in S phase. After starving in medium containing CSS for six days, the cell population distribution for LN-neo was 75% in Gl phase, 4% in G2 and 5% in S phase. In contrast, the distribution for LN-HER was 71% in Gl phase, 7% in G2 phase and 14% in S phase. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A. FCS LN-neo LN-HER l® % of Gl phase % of G2 phase % of S phase LN-neo 58 10 22 LN-HER 61 11 23 B. CSS 6D LN-neo 8 8 - j is s LN-HER % of Gl phase % of G2 phase % of S phase LN-neo 75 4 5 LN-HER 71 7 14 Chi square test, p = 0.02 126 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 5-4A. p27 expression examined by Western blot analysis in the transfected LNCaP cells. LN-neo: LNCaP transfected with parent vector. LN-HER: LNCaP transfected with recombinant HER-2/neu plasmid. FCS: cells were grown in RPMI 1640 containing 10% fetal calf serum. CSS6D: cells were grown in RPMI 1640 containing 10% charcoal stripped serum for 6 days. Top panel: Western blot of Her 2/neu expression in the LN-neo and HER-2/neu transfected cells. Middle panel: Western blot of p27 expression in the LN-neo and HER-2/neu transfected cells. In presence of FCS, LN-neo and LN-HER displayed similar p27 expression. After growth in CSS for 6 days, p27 expression increased in LN- neo cells, but not in LN-HER. Bottom panel: P-actin expression in the LN-neo and HER-2/neu transfected cells as a loading control for total proteins loaded in each lane. Figure 5-4B. The effect of DHT on p27 expression examined by Western blot in the transfected LNCaP. Top panel: Western blot of Her 2/neu expression in the LN-neo and HER-2/neu transfected cells. Second panel: Western blot of p27 expression in the LN-neo and HER-2/neu transfected cells in presence of DHT for 0 hour (CSS6D), 3, 6, 15,24, 48 hours. 127 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The expression of p27 decreased in both LN-neo and LN-HER starting at 48 hours. The optical density of p27 expression for LN-neo decreased from 12.24 at CSS 6D to 10.01 at DHT 48 hours. The same change was observed in LN-HER cells. The optical density of p27 expression for LN-HER decreased from 11.34 at CSS 6D to 9.94 at DHT 48 hours. Third panel: Western blot of p27 expression in the LN-neo and HER-2/neu transfected cells in presence of ethanol equivalent to DHT for 0 hour (CSS6D), 3, 6, 15, 24, 48 hours. For both LN-neo and LN-HER cells, the optical density of p27 expression showed no decrease due to ethanol in culture medium. Fourth panel: P-actin expression in the LN-neo and HER-2/neu transfected cells as a loading control for total amount of protein loaded in each lane. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (0 o u. <n o U L 0 £ til X 03 O o 03 03 o o c I U X z -J HER-2/neu actin B i to g : ?2 ' ® y c ^ z z ' IS q : £ § x S « $ u t H i s s a l U a U i f j U S t U S U l S S l i l i l s l i l ■# "•"* 6.64 3.62 5.42 7.43 3.81 UO UD 13.33 10.83 11,23 11.41 11.33 12.78 11.43 12.24 13.05 13.31 12.95 12.85 10.01 10-02 11.34 104 7 12.38 12.11 10.89 9.94 9.43 27 13.76 13.72 12.93 12,89 13.29 11.35 12.56 12.99 12.98 12.81 12.05 12.98 11.05 p27 p27 (with ethanol) 8.83 9.56 8.78 8.56 9.11 9.13 9 4 0 9.06 8.92 9.61 9.13 9.82 9.52 9 4 3 129 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 5-5 Effect of Her 2/neu overexpression on apoptosis. LN-neo: LNCaP transfected with parent vector. LN-HER: LNCaP transfected with recombinant HER- 2/neu plasmid. Cells were seeded at 5 X 103 in a T 25 flask. After 48 hours, the medium was changed to phenol free RPMI 1640 containing either 10% FCS or 10% CSS. After growth in FCS for 3 days or in CSS for 6 days, the cells were fixed and stained for apoptosis using in situ apoptosis detection kit (TUNEL assay). The apoptotic rate is defined as an average of apoptotic cells (brown spots) counted in 10 high power fields. In FCS, both LN-neo and LN-HER displayed low apoptotic rates (0.8/hpf, 0.7/hpf respectively). After growth in CSS for 6 days, the apoptotic rate was increased dramatically to 6.2/hpf in LN-neo, but it remained the same (0.4/hpf) in LN-HER. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LN-neo LN-Her • ~ - FCS hit O .S /h pt 0 . 7 , 'h p f CBS 65 4 ^ J W S.2 ,'hpt t t e s t , p»i0.02 C 4 .hpf 131 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 5-6. Bcl-2 and Akt expression examined by Western blot analysis in transfected LNCaP cells. Top panel: Her 2/neu expression in the LN-neo (LN-neo) and HER-2/neu transfected cells (LN-HER). FCS: cells were grown in RPMI 1640 containing 10% fetal calf serum. CSS6D: cells were grown in RPMI 1640 containing 10% charcoal stripped serum for 6 days Second panel: Bcl-2 expression in the LN-neo and HER-2/neu transfected cells. HER 2/neu transfected cells showed substantially higher Bcl-2 expression than LN-neo cells in both conditions. The optical densities of Bcl-2 expression in LN- neo were 0.04 in FCS and 0.41 in CSS; while the optical densities in LN-HER were 7.16 in FCS and 6.91 in CSS. Third panel: phospho-Akt (Ser 473) expression in the LN-neo and HER-2/neu transfected cells. HER 2/neu transfected cells showed higher phospho-Akt expression than LN-neo cells in both conditions. The optical densities of phospho-Akt expression in LN-neo were undetectable (UD) in FCS, and 0.36 in CSS. The optical densities in LN-HER were 5.72 in FCS, and 6.80 in CSS. Bottom panel: P-actin expression in the LN-neo and HER-2/neu transfected cells as a loading control for total amount of protein loaded in each lane. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Her 2/neu Bcl-2 A c t i n 7M iJi ? . § ? mi m mm «ji iiz.ii ;.u .: r . l b j . ; i 6 * ' Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 5-7A. PSA expression examined by Western blot analysis in transfected LNCaP cells. Top panel: Her 2/neu expression in the LN-neo and HER-2/neu transfected cells. FCS: cells grown in RPMI 1640 containing 10% fetal calf serum. CSS6D: cells grown in RPMI 1640 containing 10% charcoal stripped serum for 6 days. Middle panel: PSA expression in the LN-neo and HER-2/neu transfected cells. HER-2 transfected cells showed higher PSA expression than LN-neo cells in both conditions. In presence ofFCS, the optical density of PSA expression for LN-neo was 14.51 versus 21.23 for LN-HER. After starving for 6 days in CSS, the optical density of PSA expression for LN-neo was undetectable (UD) versus 1.06 for LN-HER. This result indicates that Her 2/neu overexpression activates PSA transcription in the absence of androgen. Bottom panel: P-actin expression in the LN-neo and HER-2/neu transfected cells as a loading control for total amount of protein loaded in each lane. Figure 5-7B. The effect of DHT on PSA expression examined by Western blot analysis in the transfected LNCaP. Top panel: Her 2/neu expression in the LN-neo and HER-2/neu transfected cells. Second panel: PSA expression in the LN-neo and HER-2/neu transfected cells in presence of DHT for 0 hour (CSS6D), 3, 6, 15, 24, 48 hours. PSA expression was 134 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. observed to increase in both cell lines, but the obvious rise in PSA expression in LN-HER cells occurred earlier (DHT 15hr) than LN-neo cells (DHT 24hr). Third panel: PSA expression in the LN-neo and HER-2/neu transfected cells in presence of ethanol equivalent in volume to DHT for 0 hour (CSS6D), 3, 6, 15, 24, 48 hours. As expected, for both LN-neo and LN-HER cells, the optical density of PSA expression showed no increase after adding ethanol in the culture medium. The increase of PSA expression seen in the second panel was induced by DHT added in the culture medium. Fourth panel: P-actin expression in the LN-neo and HER-2/neu transfected cells as a loading control for total amount of protein loaded in each lane. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A • i 4RI um-2(mu • psa w w w w ^ W P B p e flip p p i. AC-tgP 6 M g lW S M W Jte tta 136 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 5-8. The effect of androgen deprivation and DHT on Her 2/neu expression examined by Western blot analysis in transfected LNCaP cells. Top panel: Her 2/neu expression in the LN-neo LNCaP in presence of DHT for 0 hour (CSS6D), 3, 6, 15, 24, 48 hours. Using Her 2/neu expression in FCS as a baseline, Her 2/neu expression in LNCaP was seen to increase about 35% when the cells were starved in CSS for 6 days (Figure 5-8 A). The optical density of Her 2/neu expression was 7.61 at FCS versus 10.90 at CSS 6D. When 10 nM DHT was added back into the medium of the cells after 6 days, Her 2/neu expression decreased from 10.90 at CSS 6D to 7.73 at DHT 48 hours Second panel: Control Her 2/neu expression in the LN-neo and HER-2/neu transfected cells in presence of ethanol in volume equivalent to DHT for 0 hour (CSS6D), 3, 6, 15, 24, 48 hours. As expected, the optical density of Her 2/neu expression showed no decrease after addition of ethanol in the culture medium. This result demonstrated that the decrease of Her 2/neu expression seen in the top panel was induced by DHT added in the culture medium. Third panel: P-actin expression in the LN-neo and HER-2/neu transfected cells as a loading control for total amount of protein loaded in each lane. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 8 S 8 S ® I I I s : : : e ^ ^ LL- O O O O O Q O Q A ~ «— HER-2/neu 7.61 11.24 11.68 10.00 10.80 10.SO 1 0 J6 9.63 T.73 g «■» «BI I I H I m i m m m m i HER-2/neu (ethanol) »< K 1*4% 1 ; '.3 ! 3 t : \2 t 0 1303 ‘ H i \ i Q Actin 13.21 13JB 13.24 1ZM 13.05 1 Z T I 12.74 13.11 13.31 138 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 5-9 A Model for Her 2/neu overexpression mediated pathway to androgen independence in prostate cancer. Her 2/neu overexpression in prostate cancer cells activates Akt signaling pathway, through which it promotes androgen independent growth by down-regulation of p27 expression, while also promoting androgen independent survival by up-regulation Bcl-2 expression. Finally, through Akt pathway, Her 2/neu activates AR pathway to induce expression of AR- dependent genes such as PSA. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UKR-Z/neu P1P2 Tyrosine kinase P13K PIP3 cell membrane PTKN A kt A kt P271 Hrl-2 Proliferation .Androgen Receptor PSA Survival 140 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter six: Conclusions and perspectives The primary treatment for advanced prostate cancer is hormone therapy. Nevertheless, progress to androgen independent tumors that are resistant to such therapy is a major clinical problem. The molecular mechanism leading to androgen independence is not clear. However, several lines of evidence suggest that Her 2/neu may contribute to the development of androgen independence in prostate cancer, at least in part by activating androgen receptor through PI3K/Akt or MAPK pathways [Craft et al. 1999; Yeh et al. 1999; Signoretti et al. 2000; Wen et al. 2000]. In order to further our understanding of the role of Her 2/neu in androgen independence, we have established two model systems: (a) clinical model system which includes three cohorts of prostate cancer: tumors with no exposure to hormone therapy, tumors exposed but still sensitive to hormone therapy and advanced tumors which are resistant to hormone therapy; and (b) Cell line model system which includes mock and HER-2/neu transfected LNCaP cells. Immunohistochemical study of the clinical model system shows that androgen independence is strongly associated with high levels of Her 2/neu protein, high levels of proliferative protein Ki-67 and low levels of p27 expression. Using the cell line model system, we demonstrate that under the condition of androgen deprivation, HER-2/neu transfected cells show higher proliferation rate and lower p27 expression than the LN-neo cells. Taken together, our study indicates that HER- 141 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2/neu may contribute to androgen independent growth, probably through down- regulation of p27 expression. Since cell cycle progression is a complicated cellular process that is regulated by a number of regulators, such as p21, pl6, cyclins and Cdks. It is possible that the effect of HER-2/neu on cell cycle is not limited to p27. So in this regards, we will extend our study to some other cell cycle regulators, such as p21, pl6, cyclin D and Cyclin E. Our study has also shown that besides its effect on cell cycle regulation, Her 2/neu modulates apoptosis as well. We found that androgen independent prostate cancer is strongly associated with elevated expression of apoptotic inhibitor Bcl-2. Using the cell line model system, we demonstrate that HER-2/neu transfected cells show lower apoptotic rates in presence of hormone starvation, and higher Bcl-2 expression than the mock transfected cells. These data indicate that HER-2/neu may contribute to androgen independent survival, probably through up-regulation of Bcl- 2 expression. Since apoptosis is a complicated cellular process that is regulated by a number of proteins, in the future we are going to extend this study to some other apoptosis regulators, such as Bax, Bcl-XL. Our study demonstrates that there is an elevation of phosphorylated Akt and PSA in the HER-2/neu transfected cells, indicating that high HER-2/neu expression may activate the Akt signal transduction pathway and also AR pathway. It is believed that activated Akt phosphorylates and activates androgen receptor in the absence of androgen. [Wen et al. 2000]. Therefore, Akt is a critical element in Her 142 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2/neu mediated androgen independent pathway. Since both of Akt and AR pathway are critical for proliferation and survival, we postulate that Her 2/neu may regulate p27 and Bcl-2 expression through these two pathways. The next step of this study is going to be confirming this hypothesis by blocking Akt pathway using its specific inhibitor LY294002. In this study we have found that the “cross talk” between Her 2/neu and AR pathway may be bi-directional. Her 2/neu expression is down regulated by androgen and increased after hormone starvation. This result indicates to us that hormonal therapy has dual effects on prostate cancer. On one side it kills androgen dependent tumor cells and results in tumor shrinkage; on the other side it elevates Her 2/neu and promotes the development of androgen independence. One of our future goals is to clarify the mechanism of how androgen modulates Her 2/neu expression. We hope better understanding of the interaction between these two will enable us to design new therapeutic strategies for this lethal form of prostate cancer. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. References Adnane, J., Gaudray, P., Simon, M.-P., et al. (1989). “Proto-oncogene amplification and human breast tumor phenotype.” Oncogene 4: 1389-1395. Agus, D. B., Scher, H. I., Higgins, B., et al. 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Creator Shi, Yan (author)

Core Title Molecular mechanisms of androgen independence in prostate cancer

Degree Doctor of Philosophy

Degree Program Pathobiology

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