Chawnshang Chang - US grants

The goals of this proposal are to understand the biochemical procedures by which at target organs and to provide a molecular basis for the clinical manipulation of abnormal growth of these organs. For the past three years, we have isolated and produced two very important tools [full-length of human/rat androgen receptor (AR) cDNAs and mono-/poly- specific antibodies to AR] for the study of molecular mechanism of androgen action. We also found several mutations of AR in patients with androgen insensitivity or testicular feminization (Tfm) syndrome. Based on these discoveries, we propose to test four hypotheses: Hypothesis I: Absent or defective AR may cause the Tfm syndrome. To test this hypothesis, we plan to sequence the coding region of AR gene from human and mice with Tfm syndrome. The results may allow us to detect the abnormal AR at DNA and RNA levels. Hypothesis II: Some defective AR may prevent the translocation of AR from cytoplasm to nuclei. To test this hypothesis, we plan to apply the immunocytology with microwave-fixed method to locate AR in cultured fibroblasts from Tfm patients. The results of hypotheses I and II may allow us to determine the sequences contributing to the nuclear localization process for the AR. Hypothesis III: The prostatic glands from Tfm mice may be reinduced and developed by introduction of normal AR cDNA into the urogenital sinus (UGS) of Tfm mice. To test this hypothesis, the UGS from Tfm mice will be transfected with retrovirus containing normal AR cDNA and cultured in vivo to be examined whether prostatic glands are induced. The results may demonstrate the necessary of normal AR in the mesenchyme (stroma) for the prostate development. Hypothesis IV: The Tfm syndrome in mice may be corrected by zygote microinjection (transgenic experiment) of normal AR cDNA. To test this hypothesis, the normal AR cDNA will be cloned into expression vector containing a very strong promoter (elongation factor promoter) and microinjected into the zygote of Tfm mice. The results may provide the possibility of gene therapy for the Tfm syndrome. The above studies will permit an evaluation of the role of AR in Tfm syndrome and may eventually lead to the development of new diagnostic methods and new ideas in clinical treatment of Tfm syndrome.

cell bank /registry; cancer registry /resource; blood banks; biomedical facility; human tissue;

This proposal is for the continuation of a research project in its 4th year of funding, with goals for the characterization of TR2 and TR3 orphan receptors (TR2 and TR3) in prostate cancer. TR2 and TR3 are members of the steroid receptor superfamily that were identified in the P.I.'s laboratory. Based on the past several years' research efforts from the labs of the P.I. and others, we now know TR2 and TR3 may represent master regulators in the prostate and other organs. Target genes and their hormone response elements (from DR1 to DR6) have been isolated and characterized. Data from the P.I.'s lab suggested that TR2 could modulate several important signaling pathways that may play vital roles in the prostate growth, such as (1) Retinoids- RAR/RXR-mediated apoptosis, (2) Vitamin D-VDR-mediated differentiation, and (3) irradiation-p53 -mediated apoptosis. The expression of some viruses (such as SV40 and HPV) can be regulated by TR2. The CNTF signaling pathway can also be regulated by TR2/TR3 expression. Expression of TR3 may be linked to the prostate cancer apoptosis (as demonstrated in LNCaP and PC-3 cells). Based on the above progress, we will propose the following 3 Aims to continue the characterization of TR2 and TR3 in prostate cancer cells: Aim 1, Isolation of trans-acting factors (TAFs) that control expression of TR2 and TR3; Aim 2, Isolation of cofactors (coactivators/corepressors) that can modulate the TR2/TR3 functions; and Aim 3, To study if induction of TR3 may link to the apoptosis in human prostate tumor. The success of this proposal will not only allow us to better understand the molecular mechanism of TR2/TR3, it may also help us to find new signaling pathways in the prostate that may contribute to the development of new drugs/therapies for the battle against prostate cancer.

Prostate cancer ranks first in incidence and prevalence and second as a cause of cancer-related deaths among American men. American black men suffer the highest incidence of prostate cancer in the world. Although prostate cancer appears to develop via a multistep process, little is known about the molecular alterations involved in prostate carcinogenesis. One vital factor in prostate biology and the growth control of prostate cancer is the influence of androgens. Androgenic effects are mediated through the androgen-androgen receptor (AR) complex which acts as a transcription factor regulating the target genes. Androgen-independent tumor growth accounts for the rapid progression of prostate cancer following failure of primary androgen ablation therapy. Whether a change in the molecular mechanism of AR expression can contribute to the progression of human prostate cancer to an androgen- independent state is an important question and will be the major goal of this proposal. We have found sequences within the AR gene which could be the essential elements for control of AR expression in human prostate cancer. Hence, we have proposed several specific aims to test the following three hypotheses: Hypothesis I. Could we develop an assay based on these "essential elements" to determine the hormone-responsive state of prostate cancer (androgen-dependent versus androgen-independent stages). Hypothesis II. Cis-acting elements in human AR minigene may decide the cell-specific expression of AR and the amount of trans-acting factor that bind to these cis-acting elements can also control AR-specific expression. If we can identify these cis-acting elements and trans-acting factors, we could then develop a therapeutic drug based on the above mechanisms to treat prostate cancer by preventing prostate cancer progression into androgen-independent stage. Hypothesis III. To understand more about prostate cancer etiology, we can use this mini-AR gene-ARECAT assay or cis-acting elements and/or trans- acting factors as tools to screen prostate tumors from (1) different age groups (2) different stages of prostate cancer; and (3) different races (Taiwanese/Japanese versus American white/black); (4) spontaneous prostate tumor from rats treated with different diet.

Prostate cancer (CaP) has become the most frequently diagnosed neoplasm and the second leading cause of cancer mortality in men second to lung cancer. Androgen-independent tumor growth accounts for the rapid progression of CaP following failure of primary androgen ablation therapy. whether a change in the molecular mechanism of androgen receptor (AR) function can contribute to the progression of human CaP to an androgen-independent state is an important question and will be the major goal of this proposal. It has been demonstrated that even though CaP becomes androgen-independent in advanced stages, with the immunohistochemical techniques, investigators can still detect the existence of AR in most of the CaP samples. In addition, in the human LNCaP cell line, Kokontis et al. demonstrated that the expression of AR at both the mRNA and protein levels rises when these cells are kept in a low androgen culture environment, which may mimic the clinical environment after androgen ablation therapies. As a result, these LNCaP 104-R cells become androgen hypersensitive in the regulation of target gene expression and their proliferation can be suppressed by a low level of supplemented androgen. A similar situation might account for the "Flutamide Withdrawal Syndrome" in CaP patients. These observations have led us to suspect that failure of expression of AR may account for only a small portion of patients with androgen-independent CaP and alteration of the function of AR could result in androgen-independent tumor progression. Hence, we propose several specific aims to test the following two hypotheses: Hypothesis l: We hypothesize that AR-associated proteins (ARAs) might form a complex with A-AR and result in the androgen-activation process. We propose that if the ARA proteins are absent or available in limited quantity cells, the existing AR may not function properly. We plan to use the yeast two-hybrid and far-Western hybridization systems to clone ARAs and characterize their roles in prostate androgen action. Hypothesis II: We propose that the alterations in LNCaP 104-R, LNCaP CSS5B, PC3AR7 and PCAR1O cell behavior may be the result of the change in AR to ARA ratio (based on our data that 1:3 ratio can maximize A-AR activity). To test this hypothesis, we plan to over-express the ARA candidate(s) from hypothesis l in these cells and follow any changes in cell response to androgen stimulation. If our hypothesis is correct, we will expect the cell behavior to revert to that of the original LNCaP cells, i.e., androgen stimulates cell proliferation. If this proves to be correct, then it will be possible (1) to design a diagnostic kit for ARA to monitor the status of CaP androgen dependency and (2) to develop therapeutic agents that induce more ARA expression in CaP to overcome androgen independence. As a result, ultimately, we might find a potential protocol to manage the progression of CaP in the clinic.

DESCRIPTION: One vital factor in prostate cancer (PCa) growth is androgen. Androgen effects are mediated through androgen-androgen receptor (A-AR) interaction that can then activate or repress its target genes. Based on this principle, androgen ablation therapy with various antiandrogens to treat PCa is still being used. However, many (if not all) PCa patients progress to an androgen-independent state leading to demise. Androgen is the only steroid hormone with two active forms (testosterone, T, vs. dihydrotestosterone, DHT) and all data thus far suggest only one AR gene exists. Further study of the mechanism of these two androgens will help determine how T or DHT can differentially regulate androgen target genes. This may allow us to develop a T- or DHT- specific antiandrogen that will help us to battle PCa with fewer side effects or prevent progression to an androgen-independent stage (because we only block one type of androgen without interfering with the action of the other type).

DESCRIPTION: The testicular orphan receptor 4 (TR4) was initially cloned from prostate and testes cDNA libraries by the P.I.(Chang et al, 1994). Preliminary studies indicated that the TR4 might belong to the estrogen receptor/thyroid receptor nuclear receptor subfamily due to its ability to bind to AGGTCA direct repeats. Functional analysis showed that the TR4 could suppress retinoic acid (RA)-induced transactivation, recognize a DNA promoter in HIV-I and SV40, and induce the CNTFR gene expression. The TR4 can also modulate the thyroid hormone, Vitamin D, and PPARalpha signal cascades, and exert negative activity on erythropoietin gene expression. Our preliminary data found that ligands for RXR, PPARalpha, and CNTFR could also control the expression of the TR4, providing a bi-directional feedback, control mechanism between TR4 and other signaling pathways. Based on the above data, the investigators propose the following 5 aims to further characterize the TR4 in the prostate and testis. Aim 1) Isolation of cofactors (coactivators/corepressors) that can modulate the TR4 functions. Aim 2) 5' promoter characterization and isolation of cis-acting elements that control expression of the TR4. Aim 3) Isolation of trans-acting factors (TAFs) that control expression of the TR4. Aim 4) Identification of ligands or activators for TR4. Aim 5) Study cross talk between p53 and TR4 in testes. The successful completion of this proposal may help us to better understand the molecular mechanism of the TR4 and its potential biochemical functions.

DESCRIPTION (Provided by the applicant) Androgens, acting through the androgen receptor (AR), mediate a wide spectrum of developmental and physiological processes, including the development and maintenance of the prostate. However, it has recently been shown that AR can be transcriptionally activated in vitro by 17(3-estradiol (E2) in the presence of the AR coregulator ARA70. In rodent models, neonatal exposure to exogenous estrogen results in a dose dependent alteration of adult prostate size and histology. However, targeted disruption of the estrogen receptors (ER) a and P in mice do not conclusively show a role for either ER in prostate development, potentially indicating that exogenous estrogens may be acting at least in part through AR to influence prostate growth. Estrogens and androgens are structurally similar with the major difference occurring at the C-3 position of the steroidal A-ring where dihydroxytestosterone (DHT) carries a keto group and E2 carries a phenolic hydroxyl. To investigate the amino acid residues of AR that mediate the ability of E2 to induce AR transcription, and the mechanism through which coregulators differentiate between DHT- and E2-bound AR, we propose in Specific Aim I to isolate mutations of AR that transcriptionally respond to DHT but not E2. In Specific Aim 2, we will isolate AR mutants the preferentially respond to E2. In Specific Aim 3, we will isolate specific coregulators that differentially interact with DHT- or E2-bound AR. In Specific Aim 4, we will determine the mechanism of DHT or E2 induced transcription of AR in the presence of AR coregulators. Finally, in Specific Aim 5 we will determine the effect of E2 induction of AR transcription in prostate cells. The success of this proposal will not only allow us to understand how E2 regulates AR transcription, but will also provide information on transcription by AR in response to different ligands, which may ultimately lead to novel therapeutic approaches to prostate cancer.

[unreadable] DESCRIPTION (provided by applicant): PTEN and AR are highly involved in the prostate cancer progression. The linkage between these two antagonistic factors remains unclear. PTEN has been identified as a tumor suppressor containing a dual-specificity protein tyrosine phosphatase and lipid phosphatase domain. PTEN may use its lipid phosphatase activity to negatively regulate cell growth through inhibition of general survival signaling PI3IKIAkt pathway. Several reports have also suggested the existence of PI3K/Akt-independent pathways in PTEN-mediated signaling. Androgen serves as a mitogen for AR-positive prostate cancer cell growth. AR can be activated by some kinase or growth factors in a ligand-independent manner. Invasion inhibition and growth arrest induced by PTEN may require its protein phosphatase activity in some cell lines, but its protein targets still remain largely unknown. AR is a phosphoprotein whose activity is highly regulated by alteration of phosphorylation levels. Our preliminary data found that PTEN could interact with AR in vitro and in viva and suppress AR transactivation, suggesting the existence of a new PTEN-signaling pathway in LNCaP cells. We hypothesize that in addition to the classic PTEN-PI3K-Akt pathway PTEN can also suppress AR by direct interaction with AR via either destabilization of AR protein, slowing down AR nuclear translocation, or changing AR phosphorylation status. The consequence of these events may then result in the suppression of AR-mediated transactivation and cell proliferation. Three Aims will be proposed to test this hypothesis: Aim 1: To dissect the mechanisms of how PTEN suppresses AR transactivation. (1A: by decreasing the AR expression via destabilization of AR protein, 1 B: by a slow-down of the AR nuclear translocation, or 1C: by AR dephosphorylation). Aim 2: To find the minimum interaction domain or mutated PTEN that will interrupt the PTEN suppression of AR function. Aim 3: To determine the growth arrest and cell apoptosis induced by PTEN through the inhibition of AR-mediated pathway. The success of this proposal may provide a new molecular mechanism that may link the tumor growth suppression in AR-positive prostate cancer and may also increase therapeutic approach for prostate cancer patients in the future.

DESCRIPTION (provided by applicant): The goal of our proposed research is to explore the effects of targeted disruption of the nuclear orphan receptors TR2 and TR4, as well as consequences of disruption of the two genes in combination, in order to determine the physiological roles of these receptors. We hypothesize that both TR2 and TR4 play significant roles in the regulation of developmental, physiological, and behavioral systems. To determine the specific roles of TR2 and TR4 in vivo, we will employ mouse models exhibiting ablation of either orphan receptor, as well as animals exhibiting simultaneous ablation of both receptors. Specific aim 1: Characterization of TR4 knockout/Beta-gal knockin (TR4 -/-) mice. Specific aim 2: Characterization of TR2 knockoutJBeta-gal knockin (TR2 -/-) mice. We hypothesize this receptor is more important in early stages of development, and that TR2 -/- mice may die pre- or postnatally. Mortality will be investigated by morphological and histological analyses, and animals surviving to adulthood will be assessed for growth rate and fertility. Specific aim 3: Characterization of TR2/TR4 double knockout mice. Analysis of the double knockout animals will follow the approach described in Specific Aim 2, and the phenotypes will be analyzed in comparison to TR2 knockout, TR4 knockout, and wildtype mice. Specific Aim 4: Analysis of the effects of TR4 and/or TR2 ablation on target gene regulation. TR4, TR2, and TR2/TR4 knockout animals will be tools to study the known target genes of these orphan receptors in an in vivo system, and to confirm the physiological significance of the identified regulatory pathways, and provide sources of material for the screening of novel TR2/TR4 target genes. 4a: Determination of effect of TR4 and/or TR2 target gene ablation on known target gene expression. Compare endogenous gene expression, and protein levels of TR4 and TR2 downstream targets in knockout animals versus wildtype controls. 4b: Identifiy novel genes regulated uniquely or differentially by TR4 and/or TR2 through the use of gene microarray technology, we hope to dissect the differences in target gene regulation mediated by TR4 and TR2 and, in this way, further understand the roles of these enigmatic orphan receptors in mammalian development and physiology. In summary, this proposal provides us the first opportunity to study the potential in vivo physiological roles of the TR2 and TR4 orphan nuclear receptors.

DESCRIPTION (provided by applicant): Prostate cancer is known to commonly metastasize to bone. One of the important mediators of prostate cancer cell proliferation is androgens functioning through the androgen receptor (AR). In addition to prostate cancer cell proliferation, androgens also function to regulate osteoblasts, osteoclasts, and bone resorption. It is therefore possible that the ability of prostate cancer cells to adhere to bone and alter bone turnover resulting in bone remodeling is related to the function of AR in prostate cancer cells and in the bone. AR activity in prostate cancer cells may result in the production of growth factors that stimulate bone remodeling. Similarly, AR action in osteoblasts and osteoclasts may contribute to prostate cancer proliferation or adherence at the metastatic site. To investigate the potential cooperative role of AR in prostate cancer and bone at metastatic sites, both in vitro and in vivo model systems will be used. We have previously generated mice using lox-Cre methodology that enable us to remove AR in the whole animal or in a cell-type specific manner, including the removal of AR in osteoclasts and osteoblasts. We have also generated a subline of the AR negative prostate cancer cell PC3 that expresses AR from the AR promoter. Four specific aims are proposed making use of these reagents: Specific Aim 1, to determine the role of AR in bone remodeling by prostate cancer cells in an in vitro model system; Specific Aim 2, to determine the role of AR in an in vivo model of prostate cancer metastasis; Specific Aim 3, to determine the role of osteoclast AR function in an in vitro and in vivo model of prostate cancer metastasis; and Specific Aim 4, to determine the role of osteoblast AR function in an in vitro and in vivo model of prostate cancer metastasis. The success of this proposal will allow a better understanding of the role of androgen in the metastatic spread of prostate cancer to bone and the molecular mechanism of bone remodeling by prostate cancer cells.

DESCRIPTION (provided by applicant): The overall objective of this proposal is to investigate the role of the androgen receptor (AR) in prostate cancer metastasis. Prostate cancer is the second leading cause of cancer-related death among men in the United States. Androgen action and the functional status of the AR are believed to be the important mediators of prostate cancer development. Although androgen/AR is known to promote growth of androgen-dependent prostate tumors, its function in tumor metastasis is unclear at present. A preliminary study has been made in Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mouse models of prostate cancer and discovered that ablation of the AR in prostate epithelium resulted in development of larger pelvic lymph node (PLN) metastatic tumors and higher numbers of liver metastatic foci, suggesting that AR might function as a suppressor of prostate tumor metastasis. Therefore, the proposed studies will use various ARKO models of TRAMP mice and human prostate cancer cell lines with different AR expression to investigate the suppressor role of AR in prostate tumor metastasis. Initially prostate epithelium-specific (pes) AR knockout (ARKO) (Aim 1) and inducible ARKO TRAMP mice (Aim 2) will be generated to study the effect of pes-ARKO and induction of ARKO, after prostate tumors have developed, on PLN metastatic tumor formation and growth as well as the expression levels of various metastasis-related genes. Subsequently, the effect of suppressing AR expression (with AR-specific RNA interference) in AR-positive and restoring AR expression (with functional AR cDNA) in AR-negative PLN tumor cells and various human prostate cancer cell lines (Aim 3) on their invasive properties and expression levels of metastasis-related genes will be studied in vitro as well as in bone xenografts in SCID mice. Finally, the molecular mechanisms through which AR regulates the expression levels of various metastasis-related genes will be studied (Aim 4). Therefore, the success of this application undoubtedly will uncover an alterative role of the AR as a metastasis suppressor during prostate cancer progression. The establishment of the AR function as a metastasis suppressor and understanding of how it regulates tumor metastasis-related genes, should lead to new concepts of treatment modality and molecular targeting for prostate cancer therapy. Project Narrative: Loss of Androgen Receptor Promotes Metastatic Cancer. We will study the loss of androgen receptor in prostate epithelium and the influence of this loss on prostatic metastatic tumor invasion. The success of this application undoubtedly will uncover an alterative role of the androgen receptor as a metastasis suppressor during prostate cancer progression. The establishment of the androgen receptor function as a metastasis suppressor and understanding of how it regulates tumor metastasis-related genes, should lead to new concepts of treatments and molecular targets for prostate cancer therapy.

DESCRIPTION (provided by applicant): Primary HCC is the fifth most common cancer in the world and the third most common cause of cancer mortality. The major etiological factors leading to HCC are the infection of hepatitis B and C viruses (HBV and HCV), as well as consumption of aflatoxin B1 contaminated foods. Since a gender bias exists in the HCC incidence and cancer regression the objective of this proposal is to investigate the role of the androgen receptor (AR) in the development and growth of hepatocellular carcinoma (HCC) and explore the feasibility of targeting AR for treatment of HCC. Initially, the role of AR in HCC development and growth will be studied by comparing the carcinogen, N, N-diethylnitrosamine-induced HCC development and growth in wild type, general AR knockout (G-ARKO), and liver-specific AR knockout (L-ARKO) mice through determination of liver tumor incidence, rates of change in tumor foci number and size, and histochemical comparison of growth of preneoplastic foci, tumor cell proliferation and apoptosis indexes. These studies will be extended to HBV-mediated HCC development and growth through generation of G-ARKO and L- ARKO models of mice carrying HBV genome (HBV transgenic mice). The roles of AR in modulation of liver tumor growth deduced by ablation of AR in these mice models will be further confirmed by studying the effect of downregulating the expression of AR by AR-specific small interference RNA (AR-siRNA) on the growth of primary tumor cells of wild type and HBV transgenic mice in primary cultures and tumor xenografts in nude mice. The effect of AR-siRNA will be extended to the study of AR-expressing human hepatoma cells, both HBV-positive and HBV-negative, in tissue cultures and tumor xenografts. After having established that AR modulates the development and/or growth of liver tumor cells in these studies, the molecular mechanisms of how AR influences liver cancer incidence and progression will be studied via 4 different pathways. Public Relevance Statement: The success of this application will provide useful information pertaining to not only the role of AR in HCC development and growth, but also to the applicability of targeting AR for treatment of human primary HCC and eventually lead to more effective treatment regimens for this deadly neoplastic disease.

Sex hormones are linked to regulation of immune and inflammatory responses. Castration of male mice results in expansion of B lymphocytes in the bone marrow and peripheral blood. This effect is due to reduction of male sex hormone, androgen, which acts through activation of androgen receptor (AR). Ablation of AR in mice also leads to expansion of B cells in the bone marrow and peripheral blood, suggesting that AR plays a negative regulatory role in B lymphopoiesis. The overall objective of this proposal is to elucidate the mechanism through which AR regulates B lymphopoiesis. The proposed studies will test the hypothesis that AR regulates proliferation and apoptosis of B precursor cells. Thus, ablation of AR from bonemarrow cells will increase proliferation and and resistance to apoptosis of B cells leading to B cell expansion, whereas increased B cells might result in increased production of self-reactive B cells hence autoimmunity. To test this hypothesis, stromal cell and B cell specific AR knockout (S-ARKO and B-ARKO) mice will be generated for the proposed studies. The distribution of various bone marrow B cells along the developmental pathway and their proliferating and apoptosis rates will be determined by flow cytometry. The production of autoantibody and self-reactive B cell also will be determined. The results obtained from G-ARKO and B- ARKO mice will be compared with their wild type littermates to determine the sites of AR action in B lymphopoiesis. RNA will be isolated from purified B cells of S-ARKO, B-ARKO, and wild type mice to identify the genes related to apoptosis/proliferation affected by AR ablation so that the mechanism of AR action can be elucidated. Finally, the susceptibility of S-ARKO, B-ARKO, and wild type to induction of autoimmune disease will be studies and compared. The results of the proposed studies will provide useful information toward elucidation of the role of AR and its mechanis of action in regulating B-lymphopoiesis and autoimmunity, and better understanding and treatment of autoimmune diseases.

[unreadable] DESCRIPTION (provided by applicant): Prostate cancer is currently the second leading cancer death in American men. Prostate cancer is dependent on androgen acting through the androgen receptor (AR) for growth and survival, and androgen ablation has been used for treatment of prostate cancer. Despite an initially favorable response, hormone ablation therapy eventually fails and the cancer progresses to an incurable metastatic disease. Epidemiological data regarding prostate cancer genetics indicate roles of AR mutation, as well as AR poly-glutamine (poly-Q) polymorphism, in susceptibility to prostate cancer development and response to therapy. The overall objective of this proposal is to create mouse prostate cancer models for studying the role of the AR in prostatic cancer development and metastasis. Using the Cre-loxP conditional gene knockout technology, several derivatives of TRAMP or PTEN-deficient mouse prostate cancer models with cell type-specific AR knockout (ARKO) (post-puberty epithelial, fibroblast-, and smooth muscle cell-specific ARKO), replacement of prostatic epithelial AR with AR(T857A) [murine equivalent of human AR(T877A)] mutant will be generated for in vivo study of the roles of the AR in prostate stromal cells or epithelial cells, the AR(T877A) mutation in prostate cancer initiation, progression, or metastasis. Prostate tumor initiation, progression, and/or metastasis in these models will be examined and compared with their littermates expressing wild type AR through histological analyses, immunohistochemical determination of various cellular markers, biochemical determination of the expression levels of tumor metastasis related genes and proteins, and analysis of invasive properties of their metastatic tumor. Through the differences in these parameters between the various ARKO or AR (T857A) mice and their wild type littermates, the role of the AR in each specific cell type and the AR(T877A) mutation in prostate carcinogenesis will be delineated. Information obtained from these studies not only will provide better understanding of the roles of the AR in prostate carcinogenesis, but also will be useful for designing new treatment regimen for prostate cancer in hormone refractory state. In addition, these models may be useful for testing new drugs for prostate cancer therapy. Project Narrative: New Mice Models for Studies of Androgen Receptor in Prostate Cancer [unreadable] [unreadable] We will generate various mouse models that lack the androgen receptor in individual cells of the prostate and study their influence on prostate cancer progression. Information obtained from our studies of the differences between these mice models will lead to our understanding some roles of the androgen receptor in the development of prostate cancer and also will be useful for designing new treatments for prostate cancer. In addition, these mice models may be useful for testing new drugs and treatments for prostate cancer therapy, which might eventually be used in humans. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Urinary bladder cancer (BCa) is the fourth/tenth most common cancer among men/women in the United States. It is reported that males have significantly (approximately three times) higher risk of bladder cancer than females. However, the etiology of this sex difference in incidence is unknown. Recent studies have suggested the involvement of androgens/androgen receptor (AR) signaling in BCa progression. Using AR knockout (ARKO) mice, we found that the androgen/AR signaling might play a critical role in the development of the chemical carcinogen, N-butyl-N-4-hydroxybutyl-nitrosamine (BBN), induced bladder carcinogenesis. With BBN treatment, ARKO mice, that lack AR and with undetectable androgen, did not develop bladder tumors while their wild type littermates, with functional AR, all develop bladder tumors at the age of 50 wks. Interestingly and unexpectedly, we found near 25% of ARKO mice develop BBN-induced bladder tumor after supplementation with the androgen, dihydrotestosterone (DHT), suggesting DHT is able to function through non-AR pathways to promote bladder tumor progression. This finding was further confirmed in the human AR-negative bladder cancer cell line, TCC5637, showing that addition of DHT can promote this AR-negative bladder cell growth and invasion. Preliminary data also showed DHT could enhance the pathways from G-proteins signals to ERK/MAPK-MEK signals in BCa AR- negative cells. Based on these in vitro human cell line and in vivo mice evidences, we hypothesize that DHT can function through non-AR pathways to promote BCa initiation, growth, and invasion. We will apply the following 4 aims to prove our hypothesis and dissect potential mechanisms. Aim 1: Using the Uro- SV40T-ARKO mouse model to prove DHT can go through non-AR pathways to promote BCa progression. Aim 2: Using UPII-Ha-ras-ARKO mouse model to prove DHT can go through non-AR pathways to promote BCa progression. Aim 3: Using cell transformation/tumorigenesis assays to prove DHT can go through non-AR mediated pathways to promote BCa initiation and/or progression. Aim 4: Using human AR-negative bladder cell lines to prove DHT can go through non-AR pathways to promote BCa growth and invasion and to dissect potential mechanisms. IMPACT: Currently, BCa in humans is not generally considered hormonally dependent and therefore it is not assumed that androgen deprivation therapy (ADT) with chemical or surgical castration can be an effective treatment option for BCa patients in order to repress tumor progression. The success of this proposal to prove DHT can function through non-AR pathways to promote bladder cancer initiation and progression, not only provides in vivo evidence for a novel androgen mechanism, it may also answer the puzzle for the previous failure of using ADT to suppress androgen binding to AR for the treatment of BCa patients and may provide a new preventive/therapeutic approach to suppress BCa.

DESCRIPTION (provided by applicant): It has been suggested that normal prostate development, benign prostate hyperplasia (BPH), and prostate cancer development all require androgen/androgen receptor (AR) signaling in human and rodent. There are two types of cells, epithelial and stromal cells, constituting the prostate gland structure and mediating the physiological functions. The expression of AR in both epithelial and stromal cells may play important roles in controlling the homeostasis of prostate growth. Our recent data suggested that double stromal-cre ARKO (d-ARKO) mouse prostates have a profoundly reduced stromal AR function and impact on the homeostasis of prostate epithelial cells. We hypothesize that the AR in the stromal cells could play differential roles in benign prostate hyperplasia (BPH) and carcinogenesis. The reason we would like to compare stromal AR role in the BPH vs. cancer is that it will lead to more insights how stromal AR may differentially contribute to the etiology and progression of these two most commen prostate diseases in elder men. In the prostate field, even though there are available in vitro cell lines or tissue recombinant system to study stromal AR roles in normal prostate development, the kidney capture recombination system is grown in immune deficient mice without proper prostate microenvironment and can be only studied in 4-7 weeks. Therefore there is a lack of long-term in vivo model to investigate stromal AR impact on the BPH and prostate carcinogenesis. To date, there are still no suitable preclinical mouse models to study the stromal AR function. We propose to generate the double cre-ARKO (d-ARKO) for understanding the stromal AR role in development of BPH and carcinogenesis. Three Specific Aims will be pursued. Aim 1. To study the stromal AR role in BPH by crossing stromal d-ARKO mice with prolactin transgenic mice. Aim 2. Generation of mouse models with stromal-fibroblast/smooth muscle selective double-cre AR knockout (d-ARKO) and investigation of prostate tumorigenesis and tumor progression. Aim 3. Studying target gene and molecular mechanisms of stromal AR that could affect the epithelium during development of BPH and the prostate cancer. The accomplishment of the project will help us gain insights on the role of stromal AR in the prostate homeostasis and cancerous transformation and progression, and the results could also lead to developing new alternative treatments for BPH and prostate cancer in the future. PUBLIC HEALTH RELEVANCE: To date, the epithelial androgen receptor (AR) has drawn a lot of attention in prostate carcinogenesis and there are thousands of publications focused on addressing the role of epithelial AR in prostate cancer. For the stromal AR, there is still a lack of an in vivo model to investigate its impact on the BPH and prostate carcinogenesis. The accomplishment of the studies could help to (i) uncover new insights about the cellular functions of stromal AR, (ii) explore and understand the critical roles of the stromal AR in BPH and prostate cancer initiation and progression, and (iii) could help to formulate new and alternative therapeutic strategies by selectively targeting stromal AR and its downstream pathways to battle those two important prostate diseases.