Emily A. Ricke

Steroid hormones stimulate human prostate cancer progression and metastasis

Tissue recombinants (TRs) composed of mouse urogenital mesenchyme (mUGM) plus an immortalized nontumorigenic human prostatic epithelial cell line (BPH‐1) were grown under the kidney capsule of male athymic nude mice under different hormonal conditions. The objectives were to determine temporal plasma concentrations of testosterone (T) and estradiol‐17β (E2) that elicit progression of nontumorigenic human prostatic epithelial cells in vivo. Second, to determine whether mUGM+BPH‐1 TRs in [T+E2]‐treated hosts could progress to metastases. Control mouse hosts received no exogenous hormonal support, whereas treated mice received Silastic implants containing T and E2 for 1–4 months. Plasma from hormonally treated mice contained significantly higher (p < 0.01) concentrations of T at 1 month (11.7 vs. 0.9 ng/ml). Plasma levels of E2 in steroid implanted mice were significantly higher (p <0.05) at 2 months (104.5 vs. 25.6 ng/l) and 4 months (122.8 vs. 19.2 pg/ml). Wet weights of mUGM+BPH‐1 TRs from [T+E2]‐implanted mice were significantly larger (p < 0.001) than those from untreated hosts. Untreated mUGM+BPH‐1 TRs contained a well organized differentiated epithelium surrounded by smooth muscle stroma similar to developing prostate. In [T+E2]‐implanted mice, mUGM+BPH‐1 TRs formed carcinomas that contained a fibrous connective tissue stroma permeating the tumor; smooth muscle when present was associated with vasculature. Renal lymph nodes collected from [T+E2]‐treated mice, but not untreated mice, contained metastatic carcinoma cells. Moreover, metastases could be observed at distant sites including lung and liver. Epithelial cells isolated from untreated mUGM+BPH‐1 TRs exhibited benign histology and formed small nontumorigenic grafts when subsequently transplanted into athymic nude mice. In contrast, epithelial cells isolated from mUGM+BPH‐1 tumors of [T+E2]‐treated hosts formed large tumors that grew independent of stromal and hormonal support and developed lymph node metastases. We conclude that [T+E2]‐treatment promotes prostatic cancer progression in mUGM + BPH‐1 TRs. Use of mUGM in this system will allow future studies to utilize the power of mouse genetics to identify paracrine factors involved in human prostatic carcinogenesis.

Androgen hormone action in prostatic carcinogenesis: stromal androgen receptors mediate prostate cancer progression, malignant transformation and metastasis

It has been postulated that prostatic carcinogenesis is androgen dependent and that androgens mediate their effects primarily through epithelial cells; however, definitive proof of androgen hormone action in prostate cancer (PRCA) progression is lacking. Here we demonstrate through genetic loss of function experiments that PRCA progression is androgen dependent and that androgen dependency occurs via prostatic stromal androgen receptors (AR) but not epithelial AR. Utilizing tissue recombination models of prostatic carcinogenesis, loss of AR function was evaluated by surgical castration or genetic deletion. Loss of AR function prevented prostatic carcinogenesis, malignant transformation and metastasis. Tissue-specific evaluation of androgen hormone action demonstrated that epithelial AR was not necessary for PRCA progression, whereas stromal AR was essential for PRCA progression, malignant transformation and metastasis. Stromal AR was not necessary for prostatic maintenance, suggesting that the lack of cancer progression due to stromal AR deletion was not related to altered prostatic homeostasis. Gene expression analysis identified numerous androgen-regulated stromal factors. Four candidate stromal AR-regulated genes were secreted growth factors: fibroblast growth factors-2, -7, -10 and hepatocyte growth factor which were significantly affected by androgens and anti-androgens in stromal cells grown in vitro. These data support the concept that androgens are necessary for PRCA progression and that the androgen-regulated stromal microenvironment is essential to carcinogenesis, malignant transformation and metastasis and may serve as a potential target in the prevention of PRCA.

Testosterone and 17β-Estradiol Induce Glandular Prostatic Growth, Bladder Outlet Obstruction, and Voiding Dysfunction in Male Mice

Benign prostatic hyperplasia (BPH) and bladder outlet obstruction (BOO) are common in older men and can contribute to lower urinary tract symptoms that significantly impact quality of life. Few existing models of BOO and BPH use physiological levels of hormones associated with disease progression in humans in a genetically manipulable organism. We present a model of BPH and BOO induced in mice with testosterone (T) and 17β-estradiol (E(2)). Male mice were surgically implanted with slow-releasing sc pellets containing 25 mg T and 2.5 mg E(2) (T+E(2)). After 2 and 4 months of hormone treatment, we evaluated voiding patterns and examined the gross morphology and histology of the bladder, urethra, and prostate. Mice treated with T+E(2) developed significantly larger bladders than untreated mice, consistent with BOO. Some mice treated with T+E(2) had complications in the form of bladder hypertrophy, diverticula, calculi, and eventual decompensation with hydronephrosis. Hormone treatment caused a significant decrease in the size of the urethral lumen, increased prostate mass, and increased number of prostatic ducts associated with the prostatic urethra, compared with untreated mice. Voiding dysfunction was observed in mice treated with T+E(2), who exhibited droplet voiding pattern with significantly decreased void mass, shorter void duration, and fewer sustained voids. The constellation of lower urinary tract abnormalities, including BOO, enlarged prostates, and voiding dysfunction seen in male mice treated with T+E(2) is consistent with BPH in men. This model is suitable for better understanding molecular mechanisms and for developing novel strategies to address BPH and BOO.

In Utero and Lactational TCDD Exposure Increases Susceptibility to Lower Urinary Tract Dysfunction in Adulthood

Benign prostatic hyperplasia, prostate cancer, and changes in the ratio of circulating testosterone and estradiol often occur concurrently in aging men and can lead to lower urinary tract (LUT) dysfunction. To explore the possibility of a fetal basis for the development of LUT dysfunction in adulthood, Tg(CMV-cre);Nkx3-1(+/-);Pten(fl/+) mice, which are genetically predisposed to prostate neoplasia, were exposedin uteroand during lactation to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 1 μg/kg po) or corn oil vehicle (5 ml/kg) after a single maternal dose on 13 days post coitus, and subsequently were aged without further manipulation, or at 8 weeks of age were exposed to exogenous 17 β-estradiol (2.5 mg) and testosterone (25 mg) (T+E2) via slow release subcutaneous implants.In uteroand lactational (IUL) TCDD exposure in the absence of exogenous hormone treatment reduced voiding pressure in adult mice, but otherwise had little effect on mouse LUT anatomy or function. By comparison, IUL TCDD exposure followed by exogenous hormone treatment increased relative kidney, bladder, dorsolateral prostate, and seminal vesicle weights, hydronephrosis incidence, and prostate epithelial cell proliferation, thickened prostate periductal smooth muscle, and altered prostate and bladder collagen fiber distribution. We propose a 2-hit model whereby IUL TCDD exposure sensitizes mice to exogenous-hormone-induced urinary tract dysfunction later in life.

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging.

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Expression and colocalization of β-catenin and lymphoid enhancing factor-1 in prostate cancer progression☆

The purpose of this study was to objectively investigate β-catenin and LEF1 abundance, subcellular localization, and colocalization across benign and staged prostate cancer (PCa) specimens. A tissue microarray containing tumor-adjacent histologically benign prostate tissue (BPT; n = 48 patients), high-grade prostatic intraepithelial neoplasia (HGPIN; n = 25), localized PCa (n = 42), aggressive PCa (n = 31), and metastases (n = 22) was stained using multiplexed immunohistochemistry with antibodies toward E-cadherin, β-catenin, and LEF1. Multispectral imaging was used for quantitation, and protein expression and colocalization was evaluated across PCa progression. Stromal nuclear β-catenin abundance was greater in HGPIN and PCa compared with BPT (P < .05 for both), and epithelial nuclear β-catenin abundance was lower in metastatic PCa than in BPT (P < .05 for both). Epithelial and stromal nuclear LEF1 abundance was greater in HGPIN compared with BPT, whereas epithelial nuclear LEF1 was also greater in metastases. The proportion of epithelial and stromal nuclear double-positive β-catenin(+)/LEF1(+) cells was greater in HGPIN compared with BPT. In addition, the proportion of epithelial β-catenin(+)/LEF1(+) cells was greater in localized PCa and metastases compared with BPT. A significant amount of stromal cells were positive for LEF1 but not β-catenin. β-Catenin and LEF1 abundance were negatively correlated in the epithelium (P < .0001) but not the stroma (P > .05). We conclude that β-catenin and LEF1 colocalization is increased in HGPIN and metastasis relative to BPT, suggesting a role for β-catenin/LEF1-mediated transcription in both malignant transformation and metastasis of PCa. Furthermore, our results suggest that LEF1 abundance alone is not a reliable readout for β-catenin activity in prostate tissues.

MP31-08 A TISSUE SPECIFIC ROLE FOR LIGAND INDEPENDENT ARV7 SIGNALING IN BENIGN PROSTATE PATHOGENESIS

INTRODUCTION AND OBJECTIVES: Benign prostatic hyperplasia (BPH) can be progressive and lead to acute urinary retention in aging men. Estrogens and androgens have both been implicated as causes of development and progression of BPH. The dual 5 alpha reductase inhibitor dutasteride is currently used in men with moderateto-severe symptoms of BPH and prostate enlargement. However, the effect of dutasteride on intraprostatic steroid hormones except testosterone (T) and dihydrotestosterone (DHT) has not been examined. We evaluated the effect of dutasteride on intraprostatic androgen and estrogen milieu in men with benign prostatic hyperplasia. METHODS: Fourteen men who were scheduled for Transurethral Enucleation with Bipolar (TUEB) for BPH were prospectively studied based on their tissue samples. The human prostate was homogenized and derivatized steroidal fractions were subjected to LC-MS/ MS. Immunohistochemistry was used to quantify AR and PSA expression. The androgen index as the sum of (5xDHT) þ (1xT) was estimated to assess residual androgen activity in the prostate. The procedure for this research project was approved by the Ethics Committee of our institution. Informed consent was obtained from all patients. RESULTS: In control group (n1⁄48), mean tissue DHT level was 1.7ng/g tissue, and decreased to 0.14ng/g tissue for 0.5mg dutasteride (n1⁄46, average treatment duration 6.7M, p <0.001). Intraprostatic testosterone level in control group increased from 8.2pg/g tissue to 804pg/g tissue with 0.5mg dutasteride (p < 0.001). The downstream metabolite of DHT, 5a,3a-A-diol and 5a,3b-A-diol levels were 187.1pg/g tissue and 191.0pg/g tissue, respectively for control group and 9.0pg/g tissue and 12.4pg/g tissue, respectively for dutasteride group (p <0.001 and P < 0.01, respectively). The conversion of A-dione to 5a-A-dione is also catalyzed by 5alpha reductase in prostate and decreases in intraprostatic 5a-A-dione levels, and reciprocal rises of prostatic A-dione levels after dutasteride treatment were found (p < 0.001 and p < 0.01, respectively). There was no difference between the control and dutasteride group in terms of tissue DHEA (p 1⁄4 0.372), D5-A-diol (p 1⁄4 0.816), estrone (p 1⁄4 0.393) and estradiol (p 1⁄4 0.627). In dutasteride group, androgen index decreased by 82%, but the continued expression of AR and PSA was observed. CONCLUSIONS: Dutasteride provided near-complete suppression of intraprostatic DHT and 5a-A-dione but AR signaling pathway remain activated. In this study, increased T levels by dutasteride have no influence on estrogen levels in prostate.