Shutsung Liao - US grants

The overall objective of this project is to understand the biochemical processes of androgen actions in accessory genital organs, especially the rat prostate. The specific aim of this project has been and will be to purify and characterize: (a) the androgen receptor, (b) the nuclear acceptor protein that promote the receptor interaction with chromatin, (c) alpha-protein, a major secretory protein, and (d) an androgen sensitive specific spermine-binding protein. Alpha-protein and the spermine-binding protein have been purified to homogeneous forms and their partial amino acid sequences have been determined. The roles of some of these proteins in the intracellular regulation of the receptor activity in the prostate will be investigated further.

The long term goal of this research is to understand the structure and function of androgen receptors in the male hormone sensitive organs and cells. The specific aims in the next several years are: I. Preparation of specific anti-androgen receptor antibodies by lymphocyte transformation and hybridoma technology and/or by epitope selection methods. II. Cloning of androgen receptor cDNA by using expression libraries and antibody screening techniques, structural analysis of cDNA, and study of the regulation of the expression of androgen receptor gene in target organs. III. Cellular and intracellular localization, and structural analysis of androgen receptors by immunochemical analysis. Monoclonal antibodies and cDNA for androgen receptors may be used in the characterization of androgen receptors and its regulation in the abnormal organs and in the study of their roles in causing or in the progression of diseases such as benign prostate hyperplasia or prostate cancers.

The long-term goals of the research activity in our laboratory are to understand the biochemical processes by which steroid and other hormones affect target organs and to provide molecular bases for clinical manipulation of the abnormal growth of these organs. Our goals in this research project are to determine the structures of delta4-3-ketosteroid-5alpha-oxidoreductase (5alpha-reductases) in liver, prostate and other organs of the rat and human and to study the mechanisms involved in the regulation of 5alpha-reductase gene expression in normal and abnormal male hormone-sensitive organs and cells. The specific aims for the next five years are: I. Purification and amino acid sequence analysis of 5alpha- reductases. II. Preparation of polyclonal and monoclonal antibodies to 5alpha- reductases by immunization of animals and hybridoma techniques. III. Cloning of 5alpha-reductase cDNAs by using expression libraries, antibodies, affinity steroid binding, and specific oligonucleotide probes. IV. Structural analysis of 5alpha-reductases by determining nucleotide sequences of cloned cDNAs and deduced amino acid sequences of 5alpha-reductases. V. The use of cDNA and RNA probes, antibodies, in situ hybridization, nuclear run-off experiments, mRNA expression in oocytes, and cell transfection, in studies of 5alpha-reductase gene structures and the regulation of the expression of 5alpha- reductase genes in normal and abnormal target organs of androgens, such as prostate and skin. Studies from this project may provide new information and methods that are useful in the investigation of the regulatory mechanisms involved in the growth of androgen-sensitive organs and in the studies of molecular factors that can control 5alpha- dihydrotestosterone or 5alpha-reductase-linked androgen-insensitive syndromes, benign prostate hyperplasia, prostate cancers, acne, male-pattern baldness, and female hirsutism. Some of these biological systems will be investigated during this project period.

It is becoming clear that the mechanism of prostatic carcinogenesis and tumorigenesis is a multistep process involving a progression from pre-cancerous cells to cells which proliferate and metastasize and also a transformation from androgen-sensitive cancer to androgen-insensitive cancer. Understanding the molecular events driving the progression and transformation is critical to the early detection and method of treatment of prostate cancer. The proposed project is directed toward understanding whether and how oncogenetic events or other factors are involved in the progression and/or transformation of prostatic cancer cells. The role and function of androgen receptors in these processes will be explored. In addition, we will employ transgenic techniques to develop animal models that can be useful in the study of various aspects of prostate carcinogenesis and in the development of methods for the control of prostate cancer. Specific Aims are: 1) To determine the interactions of androgen receptor with cytoplasmic/nuclear factors (proto-oncoproteins) which mediate the phenomenon of androgen-dependent cell proliferation in the LNCaP human prostate carcinoma cell line. 2) To determine the cellular events which cause the transition of prostate cancer cell proliferation from androgen-dependent to androgen-independent. 3) To initiate a program to set up a transgenic mouse model of prostate cancer for the purpose of identifying factors involved in the induction and progression of human prostate cancer. An understanding of causal factors and the existence of an experimental animal model would be useful for the development of new treatments for prostate cancer.

The long term goals of our research are to understand the biochemical processes by which androgens and related hormones affect the prostate, seminal vesicles, and other target organs and also to provide molecular bases for treatment of abnormal growth of hormone sensitive and insensitive organs, such as benign prostate hyperplasia (BPH) and prostatic cancers. The specific aims for the next 5 year period are: (1). To study the structure and functions of human and rat receptors for 5-androstene-3beta, 17beta-diol (5A-diol). This study includes characterization of receptors for 5A-diol, cloning of their cDNAs, and production of poly- and monoclonal antibodies to the receptors. (2). To study orphan receptors whose cDNAs we have isolated in the past or will be found during the isolation of cDNAs for the 5A-diol receptors. Poly- and monoclonal antibodies to the orphan receptors will be produced. We will also screen potential ligands (new hormones?) for orphan receptors. (3). To analyze mutation in androgen receptor (AR) genes in androgen- sensitive and insensitive prostate cancer cells and to identif AR sequences/domain structures and cellular factors that are important in the modulation of the transcriptional activity and intracellular recycling/replenishment of AR in prostate cells. Androgens including 5A-diol has unique effects in many male and female target organs and can also regulate the growth of androgen or estrogen sensitive tumors. The study of the structures and mutations of ARs is essential in understanding the abnormality in androgen actions. Since the functions of ARs may be modulated by cellular protein factors, identification and characterization of these factors may be important in understanding wholly how gene expression is positively or negatively modulated by androgens. 5A-diol may act by binding to a new receptor. Antagonists of 5A-diol binding to the receptor may have therapeutic values in traeating cancer and other disorders associated with 5A-diol.

It is becoming clear that the mechanism of prostatic carcinogenesis and tumorigenesis is a multistep process involving a progression from pre-cancerous cells to cells which proliferate and metastasize and also a transformation from androgen-sensitive cancer to androgen-insensitive cancer. Understanding the molecular events driving the progression and transformation is critical to the early detection and method of treatment of prostate cancer. The proposed project is directed toward understanding whether and how oncogenetic events or other factors are involved in the progression and/or transformation of prostatic cancer cells. The role and function of androgen receptors in these processes will be explored. In addition, we will employ transgenic techniques to develop animal models that can be useful in the study of various aspects of prostate carcinogenesis and in the development of methods for the control of prostate cancer. Specific Aims are: 1) To determine the interactions of androgen receptor with cytoplasmic/nuclear factors (proto-oncoproteins) which mediate the phenomenon of androgen-dependent cell proliferation in the LNCaP human prostate carcinoma cell line. 2) To determine the cellular events which cause the transition of prostate cancer cell proliferation from androgen-dependent to androgen-independent. 3) To initiate a program to set up a transgenic mouse model of prostate cancer for the purpose of identifying factors involved in the induction and progression of human prostate cancer. An understanding of causal factors and the existence of an experimental animal model would be useful for the development of new treatments for prostate cancer.

A long-term goal of research activity in this laboratory is to understand the biochemical processes by which androgens affect target organs and to provide a molecular basis for clinical manipulation of the abnormal growth and function of organs, such as the prostate, a major site of both benign and malignant growth in men. The enzyme 5alpha-reductase plays an important role in androgenic effects by converting testosterone (T) to 5alpha-dihydrotestosterone (DHT), a process believed to amplify the androgenic signal. Excessive DHT or 5alpha-reductase activity has been implicated in a variety of androgen-dependent pathological conditions including benign prostatic hyperplasia (BPH), acne, alopecia, and female idiopathic hirsutism. Inhibition of 5alpha-reductase has been advocated as a method to treat these conditions. A thorough understanding of what cells express what isozymes of 5alpha-reductase, and what factors may control their expression will provide a sound basis for pharmacological intervention in androgen-dependent disorders. Research proposed in this application extends recent discoveries concerning 5alpha-reductase, in particular the observations that 5alpha-reductase is inhibited by specific unsaturated fatty acids and that different isozymes of 5alpha-reductase may have different cellular and subcellular locations. Specific aims in the next 5 years are: (1) Cells and tissues expressing specific isozymes of 5alpha-reductase will be tested in vivo and in vitro for inhibition of 5alpha-reductase by a variety of fatty acids. Any differential inhibition will be defined in relation to different 5alpha- reductase isozymes or the cellular environment in which they are expressed. (2) Metabolically stable analogs of unsaturated fatty acids will be synthesized as potential inhibitors of 5alpha-reductase. 5alpha- Reductase inhibitors in plants will be isolated and structurally defined. These new 5alpha-reductase inhibitors or their derivatives have potential clinical usefulness. (3) The intracellular location of different 5alpha- reductase isozymes will be determined using specific antibody probes. Differences in cellular location will be defined in relation to the structure of 5alpha-reductase isozymes (using chimeric 5alpha-reductase expressed in cell lines deficient in this enzyme) or their cellular environment. (4) The pattern of expression of different 5alpha-reductase isozymes in different cell types of an androgen target tissue will be determined using specific antibody and cDNA/cRNA probes. Stromal and epithelial cells will be isolated and screened for expression of specific isozymes. In situ hybridization and immunocytochemical detection may provide rapid detection of isozyme expression in tissue sections. Factors (androgens, IGF-I, etc.) that contribute to cell specific expression will be studied. (5) A cell's response to T may be dependent on the particular type of 5alpha-reductase expressed in the cell. Reporter gene expression in cells expressing specific isozymes will be analyzed. The role of 5alpha-reductase in androgen-regulated gene expression will be analyzed by RNA differential display methodology to determine if T and DHT have different effects on gene expression.

DESCRIPTION (Adapted from the Investigator's Abstract): Androgens control the development, growth and function of target tissues, such as the prostate, a major site of both benign and malignant growth in men. 5a-Reductase converts testosterone, the major circulating androgen in men, to dihydrotestosterone, a process believed to amplify the androgenic signal. Two different isozymes of 5a-reductase are present in rats, humans and other animals. The type 2 isozyme is critical for normal male development. Androgenic responses may depend on which isozyme is in a cell and this will be tested with an androgen-responsive reporter gene in cells designed to express components of this system. Testosterone and dihydrotestosterone may also control the expression of separate genes and this possibility will be investigated. Excessive dihydrotestosterone or 5a-reductase activity has been implicated in a variety of androgen-dependent pathological conditions including benign prostatic hyperplasia, acne, alopecia, and female idiopathic hirsutism. Inhibition of 5a-reductase has been proposed as a method to treat these conditions as well as a chemopreventative for prostate cancer, since the growth and function of this gland is dependent on dihydrotestosterone and most prostate cancers are initially androgen-dependent. Although a variety of synthetic 5a-reductase inhibitor have been developed, the first of these to be tested clinically (finasteride) has had limited efficacy. A variety of natural compounds that inhibit 5a-reductase in vitro have been identified. Some of these compounds are pytochemicals that are part of our diet. These compounds may modulate 5a-reductase activity in vivo and in so doing alter normal and malignant cellular growth and function. Naturally occurring 5a-reductase inhibitors and synthetic analogs will be tested in bioassays including growth and function of normal and malignant cells in culture, testosterone-induced accessory sex gland growth in rats and androgen-dependent growth of hamster sebaceous glands. The effect of these 5a-reductase inhibitors on the growth of androgen-dependent human prostate tumors xenografts in nude mice will be examined. Very little is known about the functional domains of 5a-reductase or the mechanism by which this enzyme reduces its various substrates. Development of new inhibitors of this enzyme as well as understanding the mechanism of inhibition of those inhibitors presently in hand would benefit from a more detailed picture of the steroid-binding domain of this protein. A recently developed photoaffinity-label will be used to identify amino acid residues in the steroid-binding domain of the isozymes.

DESCRIPTION (provided by applicant): Prostate cancer is the most commonly diagnosed malignancy in American men and the second leading cause of cancer death in men. Androgen deprivation therapy has been the standard treatment against hormone-dependent prostate cancer; unfortunately, androgen-independent cells usually emerge after a few years of androgen ablation therapy. Chemotherapy is often palliative at this stage, but it does not significantly increase the life span of prostate cancer patients. Because of this situation, many prostate cancer patients turn to alternative therapies to treat their disease, often without informing their physicians. In this proposal, two currently popular herbal preparations, green tea extracts and PC-SPES will be analyzed for their potential to affect the response of prostate cancer at different stages of progression to hormonal interventions, including androgens, anti-androgens, estrogens and inhibitors of 5(alpha)-reductase. In the first aim, it will be determined if green tea extracts and PC-SPES (and extracts of individual component herbs) affect the ability of androgens, anti-androgens and 5(alpha)-reductase inhibitors to control cell cycle processes and the growth of human prostate cancer LNCaP cells representing different stages of prostate cancer progression in culture and as tumor xenografts in athymic nude mice. In the second aim, it will be determined if green tea extracts and PC-SPES (and extracts of individual component herbs) affect the chemotherapeutic effects of epigallocatechin gallate on human prostate LNCaP cancer cells/tumors representing different stages of prostate cancer progression, in culture and in athymic mice. In the third aim, it will be determined if PC-SPES modulates the effects of estrogens and if estrogenic components of PC-SPES are responsible for effects on the cell cycle and the growth of prostate cancer cells representing different stages of prostate cancer progression in culture and in athymic mice.

DESCRIPTION (provided by applicant): Prostate cancer is the most commonly diagnosed malignancy in American men and is the second leading cause of cancer death among men. Androgen deprivation or antiandrogen therapy has been the frontline treatment against metastatic hormone-dependent prostate cancer. Unfortunately, androgen-independent cells almost always aggressively emerge after one to three years of androgen ablation therapy. The mechanisms by which cell cycle is controlled by androgen in prostate cancer cells, as well as the mechanisms by which androgen-dependent tumor cells escape androgenic control of cell cycle are poorly understood. Thus there is a need for a more thorough understanding of these mechanisms, as well as for innovative non-endocrine strategies to halt proliferation of hormone-independent cancer cells. We have developed a unique laboratory model of human prostate cancer progression based on the androgen-dependent human prostate cancer cell line LNCaP. We have observed that androgen-dependent LNCaP 104-S tumor cells undergo a specific G1 arrest after androgen withdrawal. Conversely, androgen-independent LNCaP 104-R1/l04-R2 cells, which are cells derived from the clonal LNCaP 104-S cell line after long-term androgen deprivation, undergo G1 arrest after androgen treatment. We have now generated fully androgen-insensitive 104-IS cells from 104-S cells by serial treatment with the antiandrogen Casodex followed by androgen. 104-IS cells may represent the final stage of the clinical progression to hormone-refractory growth. We have identified Cdk2 as the most androgen-sensitive cyclin-dependent kinase in 104-S, 104-R1 and 104-R2 cells. We have also identified the cdk inhibitor p27kip1 as a factor that is partly responsible for androgen-induced G1 arrest in 104R1 and 104-R2 cells. In the first aim of this proposal we seek to identify other components of the signaling pathway starting from androgen receptor activation and terminating at either cell cycle progression or cell cycle arrest. In the second aim we seek to determine how this signaling pathway is disrupted or bypassed in androgen-insensitive cells. In the third aim we wish to explore the use of a sterol antagonist of nuclear receptors that regulate lipid homeostasis to induce apoptosis in androgen-dependent and independent prostate cancer cells. In the fourth aim we wish to explore the use of the green tea catechin epigallocatechin gallate as an adjuvant drug in combination with androgen or antiandrogen to repress prostate tumor growth and progression.

DESCRIPTION (provided by applicant): Prostate cancer (PCa) is the most commonly diagnosed non-cutaneous cancer and is the third leading cause of cancer death in American men. As such it remains a major public health problem in the US despite improvements in diagnosis and treatment. The role of androgen receptor (AR) signaling in recurrent PCa cell growth is the subject of continuing research. It has been recognized for many years that androgen, by binding to and activating AR, promotes early stage PCa cell survival and growth. This has been the basis for androgen ablation therapy, which has served as a front-line therapy for metastatic PCa. Unfortunately, the effectiveness of androgen ablation therapy is short-lived as recurrent hormone-independent PCa cells usually emerge after one to three years of treatment. Our laboratory has also identified a repressive function of androgen in PCa cells when AR is expressed at high level. We hypothesize that this repression function of AR may be a remnant of AR action in normal healthy prostate epithelial cells and that restoring this repression function by over-expression may be effective therapy in many PCa patients. In the first aim we propose to test this hypothesis in preclinical "proof of concept" experiments by generating adenoviral constructs expressing the AR gene and use them to infect a wide array of prostate tumor cells in vitro cell culture and in vivo as tumor xenografts in athymic mice. In the second aim, will explore the mechanistic basis for androgenic repression of growth and induction of apoptosis to provide a rationale for AR replacement therapy. In the third aim we will examine novel signaling pathways that may drive cell proliferation in androgen-independent cells. We recently uncovered three regulators in a screen for differentially expressed genes in androgen-dependent vs. androgen-independent PCa cells that we hypothesize cooperate to confer androgen-independent growth. In the fourth aim, we will examine liver X receptor (LXR) signaling and the regulation of cholesterol and lipid homeostasis in PCa cells as a novel means to control cell growth. We recently found that LXR agonists repress cell proliferation in both androgen-dependent and -independent PCa cells.