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(−)-Epigallocatechin gallate (EGCG), a major tea polyphenol, elicits anticancer effects. However, the mechanism of action is not fully understood. Our laboratory previously showed that EGCG inhibits heat shock protein 90 (HSP90). We used nontumorigenic (NT), tumorigenic, and metastatic cancer cells from a novel human prostate cancer progression model to test the hypotheses that certain stages are more or less sensitive to EGCG and that sensitivity is related to HSP90 inhibition. Treatment of cells with EGCG, novobiocin, or 17-AAG resulted in more potent cytotoxic effects on tumorigenic and metastatic cells than NT cells. When tumorigenic or metastatic cells were grown in vivo, mice supplemented with 0.06% EGCG in drinking water developed significantly smaller tumors than untreated mice. Furthermore, EGCG prevented malignant transformation in vivo using the full prostate cancer model. To elucidate the mechanism of EGCG action, we performed binding assays with EGCG-Sepharose, a C-terminal HSP90 antibody, and HSP90 mutants. These experiments revealed that EGCG-Sepharose bound more HSP90 from metastatic cells compared with NT cells and binding occurred through the HSP90 C-terminus. In addition, EGCG bound HSP90 mutants that mimic both complexed and uncomplexed HSP90. Consistent with HSP90 inhibitory activity, EGCG, novobiocin, and 17-AAG induced changes in HSP90-client proteins in NT cells and larger differences in metastatic cells. These data suggest that EGCG may be efficacious for the treatment of prostate cancer because it preferentially targets cancer cells and inhibits a molecular chaperone supportive of the malignant phenotype. Cancer Prev Res; 8(3); 249–57. ©2015 AACR.

Supplemental Methods, Figure 1, Tables 1A and B. Supplemental Figure 1: Chaperone function assay; Supplemental Table 1A: Densitrometry analysis of HSP90-clients in response to EGCG; Supplemental Table 1B: Densitometry analysis of HSP90-clients in non-tumorigenic vs. metastatic cells

Objective To investigate the relationship between metabolic syndrome (MetS) and lower urinary tract symptoms (LUTS) with functional and anatomic changes of the lower urinary tract with MRI. Materials and Methods The bladder and prostate of 95 subjects (56M, 39F) were segmented on T2-weighted pelvic MRI using Materialize Mimics 3D software. Bladder wall volume (BWV), post-void residual (PVR) and prostate volume (PV) were quantified from the 3D renderings. LUTS were quantified using validated questionnaires administered at the time of MRI. Wilcoxin rank sum, win ratio and chi-square tests were used to correlate symptom scores, BWV, PVR and PV in patients 1) without vs with MetS, 2) with mild (IPSS or UDI-6: 0-7) vs moderate-severe (IPSS: 8-35 or UDI-6: ≥8) and 3) normal vs enlarged prostates (>40cm3). Multivariate linear regression was used to determine predictors for BWV, PVR and PV. Results Men with MetS had increased BWV (66.8 vs 51.1cm3, P = .003), higher PVR (69.1 vs 50.5cc, P= .05) and increased PV (67.2 vs 40.1cm3, P= .01). Women without and with MetS had similar BWV, PVR and LUTS (P= .3-.78). There was no difference in prevalence of MetS, BWV, PVR or PV in men or women with mild vs moderate-severe LUTS (P = .26-.97). Men with enlarged prostates were more likely to have MetS (P = .003). There was no difference in BWV, PVR and LUTS for men with normal vs enlarged prostates (P= .44-.94). In men, BWV was highly correlated with MetS (P = .005) on regression analysis. Conclusion MetS leads to detrusor hypertrophy and may contribute to impaired bladder function, likely related to the effect on the prostate.

Benign Prostatic Hyperplasia (BPH) occurs progressively with aging in men and drives deteriorating symptoms collectively known as Lower Urinary Tract Symptoms (LUTS). Age associated changes in circulating steroid hormones, and prostate inflammation have been postulated in the etiology of BPH/LUTS. The link between hormones and inflammation in the development of BPH/LUTS is conflicting because they may occur independently or as sequential steps in disease pathogenesis. This study aimed to decipher the prostatic immune landscape in a mouse model of lower urinary tract dysfunction (LUTD). Steroid hormone imbalance was generated by the surgical implantation of testosterone (T) and estradiol (E2) pellets to male C57BL/6J mice and gene expression analysis was performed on ventral prostates (VP). These experiments identified an increase in the expression of macrophage markers andSpp1/osteopontin (OPN). Localization studies of OPN pinpointed that OPN+ macrophages travel to the prostate lumen and transition into lipid accumulating foam cells. We also observed a significantly increased number of tissue macrophages in the VP which was prevented in OPN knockout (OPN-KO) mice. In contrast, mast cells, but not macrophages, were significantly elevated in the dorsal prostate of T+E2 treated mice which was diminished in OPN-KO mice. Steroid hormone implantation progressively increased urinary frequency, which was ameliorated in OPN-KO mice. Our study underscores the role of age associated steroid hormone imbalances as a mechanism of expanding the prostatic macrophage population, their luminal translocation and foam cell differentiation.

Mass spectrometry-based discovery glycoproteomics is highly dependent on the use of chromatography paradigms amenable to analyte retention and separation. When compared against established stationary phases such as reversed-phase and hydrophilic interaction liquid chromatography, reports utilizing porous graphitic carbon have detailed its numerous advantages. Recent efforts have highlighted the utility in porous graphitic carbon in high-throughput glycoproteomics, principally through enhanced profiling depth and liquid-phase resolution at higher column temperatures. However, increasing column temperature has been shown to impart disparaging effects in glycopeptide identification. Herein we further elucidate this trend, describing qualitative and semiquantitative effects of increased column temperature on glycopeptide identification rates, signal intensity, resolution, and spectral count linear response. Through analysis of enriched bovine and human glycopeptides, species with high mannose and sialylated glycans were shown to most significantly benefit and suffer from high column temperatures, respectively. These results provide insight as to how porous graphitic carbon separations may be appropriately leveraged for glycopeptide identification while raising concerns over quantitative and semiquantitative label-free comparisons as the temperature changes. RAW MS glycoproteomic data are available via ProteomeXchange with identifier PXD034354.

Innate immune cell infiltration into neoplastic tissue is the first line of defense against cancer and can play a deterministic role in tumor progression. Here, we describe a series of assays, using a reconfigurable microscale assay platform (i.e. Stacks), which allows the study of immune cell infiltration in vitro with spatiotemporal manipulations. We assembled Stacks assays to investigate tumor–monocyte interactions, re-education of activated macrophages, and neutrophil infiltration. For the first time in vitro, the Stacks infiltration assays reveal that primary tumor-associated fibroblasts from specific patients differ from that associated with the benign region of the prostate in their ability to limit neutrophil infiltration as well as facilitate monocyte adhesion and anti-inflammatory monocyte polarization. These results show that fibroblasts play a regulatory role in immune cell infiltration and that Stacks has the potential to predict individual patients’ cancer-immune response.

Highlights • BPA results shown in publications from independent labs were repeated in a guideline study. • BPA had effects at 2.5 ug/kg/day in the brain, prostate, urinary tract, ovary, mammary gland, heart. • In mammary gland, prostate stem cell numbers, ovary a non-monotonic BPA dose-response was observed. • 2.5 ug/kg/day should be the new LOAEL for BPA., “Consortium Linking Academic and Regulatory Insights on BPA Toxicity” (CLARITY-BPA) was a comprehensive “industry-standard” Good Laboratory Practice (GLP)-compliant 2-year chronic exposure study of bisphenol A (BPA) toxicity that was supplemented by hypothesis-driven independent investigator-initiated studies. The investigator-initiated studies were focused on integrating disease-associated, molecular, and physiological endpoints previously found by academic scientists into an industry standard guideline-compliant toxicity study. Thus, the goal of this collaboration was to provide a more comprehensive dataset upon which to base safety standards and to determine whether industry-standard tests are as sensitive and predictive as molecular and disease-associated endpoints. The goal of this report is to integrate the findings from the investigator-initiated studies into a comprehensive overview of the observed impacts of BPA across the multiple organs and systems analyzed. For each organ system, we provide the rationale for the study, an overview of methodology, and summarize major findings. We then compare the results of the CLARITY-BPA studies across organ systems with the results of previous peer-reviewed studies from independent labs. Finally, we discuss potential influences that contributed to differences between studies. Developmental exposure to BPA can lead to adverse effects in multiple organs systems, including the brain, prostate gland, urinary tract, ovary, mammary gland, and heart. As published previously, many effects were at the lowest dose tested, 2.5μg/kg /day, and many of the responses were non-monotonic. Because the low dose of BPA affected endpoints in the same animals across organs evaluated in different labs, we conclude that these are biologically – and toxicologically – relevant.

Prostate cancer (CaP) driven by androgen receptor (AR) is treated with androgen deprivation; however, therapy failure results in lethal castration-resistant prostate cancer (CRPC). AR-low/negative (ARL/−) CRPC subtypes have recently been characterized and cannot be targeted by hormonal therapies, resulting in poor prognosis. RNA-binding protein (RBP)/helicase DDX3 (DEAD-box helicase 3 X-linked) is a key component of stress granules (SG) and is postulated to affect protein translation. Here, we investigated DDX3-mediated posttranscriptional regulation of AR mRNA (messenger RNA) in CRPC. Using patient samples and preclinical models, we objectively quantified DDX3 and AR expression in ARL/− CRPC. We utilized CRPC models to identify DDX3:AR mRNA complexes by RNA immunoprecipitation, assess the effects of DDX3 gain/loss-of-function on AR expression and signaling, and address clinical implications of targeting DDX3 by assessing sensitivity to AR-signaling inhibitors (ARSI) in CRPC xenografts in vivo. ARL/− CRPC expressed abundant AR mRNA despite diminished levels of AR protein. DDX3 protein was highly expressed in ARL/− CRPC, where it bound to AR mRNA. Consistent with a repressive regulatory role, DDX3 localized to cytoplasmic puncta with SG marker PABP1 in CRPC. While induction of DDX3-nucleated SGs resulted in decreased AR protein expression, inhibiting DDX3 was sufficient to restore 1) AR protein expression, 2) AR signaling, and 3) sensitivity to ARSI in vitro and in vivo. Our findings implicate the RBP protein DDX3 as a mechanism of posttranscriptional regulation for AR in CRPC. Clinically, DDX3 may be targetable for sensitizing ARL/− CRPC to AR-directed therapies.

Lower urinary tract symptoms (LUTS) is common in aging males. Disease etiology is largely unknown but likely includes inflammation and age-related changes in steroid hormones. Diagnosis is currently based on subjective symptom scores, and mainstay treatments can be ineffective and bothersome. Biomarker discovery efforts could facilitate objective diagnostic criteria for personalized medicine and new potential druggable pathways. To identify urine metabolite markers specific to hormone-induced bladder outlet obstruction, we applied our custom synthesized multiplex isobaric tags to monitor the development of bladder outlet obstruction across time in an experimental mouse model of LUTS. Mouse urine samples were collected before treatment and after 2, 4, and 8 weeks of steroid hormone treatment and subsequently analyzed by nanoflow ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry. Accurate and high-throughput quantification of amine-containing metabolites was achieved by 12-plex DiLeu isobaric labeling. Metandem, a novel online software tool for large-scale isobaric labeling-based metabolomics, was used for identification and relative quantification of labeled metabolites. A total of 59 amine-containing metabolites were identified and quantified, 9 of which were changed significantly by the hormone treatment. Metabolic pathway analyses showed that three metabolic pathways were potentially disrupted. Among them, the arginine and proline metabolism pathway was significantly dysregulated both in this model and in a prior analysis of LUTS patient samples. Proline and citrulline were significantly changed in both samples and serve as attractive candidate biomarkers. The 12-plex DiLeu isobaric labeling with Metandem data processing presents an accessible and efficient workflow for an amine-containing metabolome study in biological specimens.

The National Institutes of Health leveled new focus on sex as a biological variable with the goal of understanding sex-specific differences in health and physiology. We previously published a functional assessment of the impact of sex, androgens, and prostate size on C57BL/6J mouse urinary physiology (Ruetten H, Wegner KA, Zhang HL, Wang P, Sandhu J, Sandhu S, Mueller B, Wang Z, Macoska J, Peterson RE, Bjorling DE, Ricke WA, Marker PC, Vezina CM. Am J Physiol Renal Physiol 317: F996–F1009, 2019). Here, we measured and compared five characteristics of urethral histology (urethral lumen diameter and area, epithelial cell count, epithelial and rhabdosphincter thickness, epithelial cell area, and total urethral area) in male and female 9-wk-old C57BL/6J mice using hematoxylin and eosin staining. We also compared male mice with castrated male mice, male and female mice treated with the steroid 5α-reductase inhibitor finasteride or testosterone, or male mice harboring alleles ( Pbsn4cre/+; R26RDta/+) that reduce prostate lobe mass. The three methods used to reduce prostate mass (castration, finasteride, and Pbsn4cre/+; R26RDta/+) changed urethral histology, but none feminized male urethral histology (increased urethral epithelial area). Exogenous testosterone caused increased epithelial cell count in intact females but did not masculinize female urethral histology (decrease epithelial area). Our results lay a critical foundation for future studies as we begin to parse out the influence of hormones and cellular morphology on male and female urinary function.

Benign prostatic hyperplasia and related lower urinary tract symptoms remain common, costly, and impactful issues for aging males. The etiology and pathogenesis are multifactorial and include steroid hormone changes and inflammation. Noninvasive markers could one day inform personalized medicine, but interindividual variation and lack of healthy age-matched controls hamper research. Experimental models are appealing for insight into disease mechanisms. Here, we present a spatiotemporal proteomics study in a mouse model of hormone-induced urinary dysfunction. Urine samples were collected noninvasively across time: before, during, and after disease onset. A microcomputed tomography analysis implicated the prostate as a spatially relevant contributor to bladder outlet obstruction. Prostates were collected after disease onset and compared with control mice. Notable changes in urine include proteins representing oxidative stress defense and acute phase inflammatory response processes. In the prostate, hormone treatment led to perturbations related to an oxidative stress response and H2O2 metabolism. Several protein changes coincided in both urine and the prostate tissue, including glutathione peroxidase 3, glutathione hydrolase 1 proenzyme, and vitamin D-binding protein. This study supports the concept of noninvasive urinary biomarkers for prostate disease diagnostics. Oxidative stress and acute phase inflammatory processes were identified as key consequences of hormone-induced bladder outlet obstruction. Future research into antioxidants and anti-inflammatories in prostate diseases appears promising.

Bisphenol A (BPA) is a contaminant in virtually all Americans. To examine BPA's adverse effects, the FDA-NCTR, NIEHS, and 14 groups of academic scientists formed a consortium: CLARITY-BPA. The purpose of our study was to investigate the effects of a wide range of doses of BPA on fetal development of the NCTR CD-SD male rat urogenital sinus (UGS). Pregnant rats were administered BPA or positive control ethinylestradiol (EE2) daily, via oral gavage, from gestational day 6 through parturition. Tissues were collected on postnatal day 1 and the UGS was analyzed using computer-assisted 3-D reconstruction. Importantly, only low doses of BPA, as well as EE2, significantly changed birth weight and UGS morphology, including an increased size of the colliculus and decreased size of the urethra, consistent with prior reported BPA and EE2 effects. Our findings provide further evidence that BPA mediates nonmonotonic developmental effects on the fetal urogenital sinus.

Continual development in instrumental and analytical techniques have aided in establishing rigorous connections between protein glycosylation and human illness. These illnesses, such as various forms of cancer, are often associated with poor prognoses, prompting the need for more comprehensive characterization of the glycoproteome. While innovative instrumental and computational strategies have largely benefited glycoproteomic analyses, less attention is given to benefits gained through alternative, optimized chromatographic techniques. Porous graphitic carbon (PGC) chromatography has gained considerable interest in glycomics research due to its mobile phase flexibility, increased retention of polar analytes and improved structural elucidation at higher temperatures. PGC has yet to be systematically compared against or in tandem with standard reversed phase liquid chromatography (RPLC) in high-throughput bottom-up glycoproteomics experiments, leaving the potential benefits unexplored. Performing comparative analysis of single and biphasic separation regimes at a range of column temperatures illustrates complementary advantages for each method. PGC separation is shown to selectively retain shorter, more hydrophilic glycopeptide species, imparting higher average charge, and exhibiting greater microheterogeneity coverage for identified glycosites. Additionally, we demonstrate that liquid-phase separation of glycopeptide isomers may be achieved through both single and biphasic PGC separations, providing a means towards facile, multidimensional glycopeptide characterization. Beyond this, we demonstrate how utilization of multiple separation regimes and column temperatures can aid in profiling the glycoproteome in tumorigenic and aggressive prostate cancer cells. RAW MS proteomics and glycoproteomics datasets have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD024196 (10.6019/PXD024196) and PXD024195, respectively.

Fibrogenic and inflammatory processes in the prostate are linked to the development of lower urinary tract symptoms (LUTS) in men. Our previous studies identified that osteopontin (OPN), a pro-fibrotic cytokine, is abundant in the prostate of men with LUTS, and its secretion is stimulated by inflammatory cytokines potentially to drive fibrosis. This study investigates whether the lack of OPN ameliorates inflammation and fibrosis in the mouse prostate. We instilled uropathogenic E. coli (UTI89) or saline (control) transurethrally to C57BL/6J (WT) or Spp1tm1Blh/J (OPN-KO) mice and collected the prostates one or 8 weeks later. We found that OPN mRNA and protein expression were significantly induced by E. coli-instillation in the dorsal prostate (DP) after one week in WT mice. Deficiency in OPN expression led to decreased inflammation and fibrosis and the prevention of urinary dysfunction after 8 weeks. RNAseq analysis identified that E. coli-instilled WT mice expressed increased levels of inflammatory and fibrotic marker RNAs compared to OPN-KO mice including Col3a1, Dpt, Lum and Mmp3 which were confirmed by RNAscope. Our results indicate that OPN is induced by inflammation and prolongs the inflammatory state, genetic blockade of OPN accelerates recovery after inflammation, including a resolution of prostate fibrosis.

Key Points Question Is frailty associated with increased risk of progression of benign prostatic hyperplasia (BPH) symptoms and serious adverse events among men receiving drug therapy? Findings In a cohort study of 3047 men with moderate-to-severe lower urinary tract symptoms due to suspected BPH, higher deficit accumulation frailty index was associated with a higher risk of clinical BPH progression, particularly among men receiving doxazosin with finasteride, and serious adverse events. Meaning These findings suggest that frail men with lower urinary tract symptoms due to suspected BPH are more likely to have progression of symptoms, despite drug therapy, as well as serious adverse events., This cohort study examines the association between a deficit accumulation frailty index and clinical progression or serious adverse events among men using drug therapy for benign prostatic hyperplasia (BPH)., Importance Benign prostatic hyperplasia (BPH) in older men can cause lower urinary tract symptoms (LUTS), which are increasingly managed with medications. Frailty may contribute to both symptom progression and serious adverse events (SAEs), shifting the balance of benefits and harms of drug therapy. Objective To assess the association between a deficit accumulation frailty index and clinical BPH progression or SAE. Design, Setting, and Participants This cohort study used data from the Medical Therapy of Prostatic Symptoms trial, which compared placebo, doxazosin, finasteride, and combination therapy in men with moderate-to-severe LUTS, reduced urinary flow rate, and no prior BPH interventions, hypotension, or elevated prostate-specific antigen. Enrollment was from 1995 to 1998, and follow-up was through 2001. Data were assessed in February 2021. Exposures A frailty index (score range, 0-1) using 68 potential deficits collected at baseline was used to categorized men as robust (score ≤0.1), prefrail (score 0.1 to

INTRODUCTION AND OBJECTIVE:Fibrogenic and inflammatory processes in the prostate are linked to the development of lower urinary tract symptoms (LUTS) in men, but the molecular mechanism is unknown....

The void spot assay (VSA) is a cost‐effective method for evaluating and quantifying mouse urinary voiding phenotypes. The VSA has been used to differentiate voiding behaviors between experimental groups, but not as a diagnostic assay. To build toward this goal, we used the VSA to define voiding patterns of male mice with diabetic diuresis (BTBR.Cg‐Lep ob /WiscJ mice), irritative urinary dysfunction (E. coli UTI89 urinary tract infection), and obstructive urinary dysfunction (testosterone and estradiol slow‐release implants) compared to their respective controls. Many studies compare individual VSA endpoints (urine spot size, quantity, or distribution) between experimental groups. Here, we consider all endpoints collectively to establish VSA phenomes of mice with three different etiologies of voiding dysfunction. We created an approach called normalized endpoint work through (NEW) to normalize VSA outputs to control mice, and then applied principal components analysis and hierarchical clustering to 12 equally weighted, normalized, scaled, and zero‐centered VSA outcomes collected from each mouse (the VSA phenome). This approach accurately classifies mice based on voiding dysfunction etiology. We used principal components analysis and hierarchical clustering to show that some aged mice (>24 m old) develop an obstructive or a diabetic diuresis VSA phenotype while others develop a unique phenotype that does not cluster with that of diabetic, infected, or obstructed mice. These findings support use of the VSA to identify specific urinary phenotypes in mice and the continued use of aged mice as they develop urinary dysfunction representative of the various etiologies of LUTS in men., Our findings support continued use of the void spot assay for etiologic and therapeutic testing in mice and demonstrates for the first time that the void spot assay is not only sensitive enough to detect differences from controls but also separate different types of voiding dysfunction. Our findings also suggest that aged mice develop urinary dysfunction consistent with the range of etiologies in men and supports their use as an ideal animal model in LUTS research. This work opens the door to future studies combining VSA data with other anatomical and physiological endpoints, increasing the power of future multivariate analysis, and facilitating model building for diagnostic urinary physiology assays in mice.

Fibrogenic and inflammatory processes in the prostate are linked to the development of lower urinary tract symptoms (LUTS) in men. Our previous studies identified that osteopontin (OPN), a pro-fibrotic cytokine, is abundant in the prostate of men with LUTS and its secretion is stimulated by inflammatory cytokines potentially to drive fibrosis. This study investigates whether the lack of OPN ameliorates inflammation and fibrosis in the mouse prostate.We instilled uropathogenic E. coli (UTI89) or saline (control) transurethrally to C57BL/6J (WT) or Spp1tm1Blh/J (OPN-KO) mice and collected the prostates one or 8 weeks later. We found that OPN mRNA and protein expression were significantly induced by E. coli-instillation in the dorsal prostate (DP) after one week in WT mice. Deficiency in OPN expression led to decreased inflammation and fibrosis and the prevention of urinary dysfunction after 8 weeks. RNAseq analysis identified that E. coli-instilled WT mice expressed increased levels of inflammatory and fibrotic marker RNAs compared to OPN-KO mice including Col3a1, Dpt, Lum and Mmp3 which were confirmed by RNAscope.Our results indicate that OPN is induced by inflammation and prolongs the inflammatory state; genetic blockade of OPN accelerates recovery after inflammation, including a resolution of prostate fibrosis.

Benign prostatic hyperplasia/lower urinary tract dysfunction (LUTD) affects nearly all men. Symptoms typically present in the fifth or sixth decade and progressively worsen over the remainder of life. Here, we identify a surprising origin of this disease that traces back to the intrauterine environment of the developing male, challenging paradigms about when this disease process begins. We delivered a single dose of a widespread environmental contaminant present in the serum of most Americans [2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD), 1 µg/kg], and representative of a broader class of environmental contaminants, to pregnant mice and observed an increase in the abundance of a neurotrophic factor, artemin, in the developing mouse prostate. Artemin is required for noradrenergic axon recruitment across multiple tissues, and TCDD rapidly increases prostatic noradrenergic axon density in the male fetus. The hyperinnervation persists into adulthood, when it is coupled to autonomic hyperactivity of prostatic smooth muscle and abnormal urinary function, including increased urinary frequency. We offer new evidence that prostate neuroanatomical development is malleable and that intrauterine chemical exposures can permanently reprogram prostate neuromuscular function to cause male LUTD in adulthood., Summary: We describe a new mechanism of benign prostate disease, initiated by fetal chemical exposure, which durably increases prostatic noradrenergic axon density and causes smooth muscle hyperactivity and urinary voiding dysfunction.

The purpose of this symposium report is to summarize information from a session 3 oral presentation at the Society of Toxicologic Pathology Annual Symposium in Raleigh, North Carolina. Mice are genetically tractable and are likely to play an important role in elucidating environmental, genetic, and aging-related mechanisms of urinary dysfunction in men. We and others have made significant strides in developing quantitative methods for assessing mouse urinary function and our collaborators recently showed that aging male mice, like men, develop urinary dysfunction. Yet, it remains unclear how mouse prostate anatomy and histology relate to urinary function. The purpose of this report is to share foundational resources for evaluating mouse prostate histology and urinary physiology from our recent publication “Impact of Sex, Androgens, and Prostate Size on C57BL/6J Mouse Urinary Physiology: Functional Assessment.” We will begin with a review of prostatic embryology in men and mice, then move to comparative histology resources, and conclude with quantitative measures of rodent urinary physiology.

Laboratory mice are used to identify causes of urinary dysfunction including prostate-related mechanisms of lower urinary tract symptoms. Effective use of mice for this purpose requires a clear understanding of molecular, cellular, anatomic, and endocrine contributions to voiding function. Whether the prostate influences baseline voiding function has not been specifically evaluated, in part because most methods that alter prostate mass also change circulating testosterone concentrations. We performed void spot assay and cystometry to establish a multiparameter “baseline” of voiding function in intact male and female 9-wk-old (adult) C57BL/6J mice. We then compared voiding function in intact male mice to that of castrated male mice, male (and female) mice treated with the steroid 5α-reductase inhibitor finasteride, or male mice harboring alleles ( Pbsn4cre/+; R26RDta/+) that significantly reduce prostate lobe mass by depleting prostatic luminal epithelial cells. We evaluated aging-related changes in male urinary voiding. We also treated intact male, castrate male, and female mice with exogenous testosterone to determine the influence of androgen on voiding function. The three methods used to reduce prostate mass (castration, finasteride, and Pbsn4cre/+; R26RDta/+) changed voiding function from baseline but in a nonuniform manner. Castration feminized some aspects of male urinary physiology (making them more like intact female mice) while exogenous testosterone masculinized some aspects of female urinary physiology (making them more like intact male mice). Our results provide evidence that circulating testosterone is responsible in part for baseline sex differences in C57BL/6J mouse voiding function while prostate lobe mass in young, healthy adult mice has a lesser influence.

Survival rates decrease significantly when localized prostate cancer (CaP) becomes metastatic, emphasizing the need for improved targeted therapies. DDX3, an RNA helicase, has widespread functions in RNA regulation, in both the nucleus and cytoplasm. Although DDX3 has been implicated as a prognostic marker for many cancers, including primary CaP, its expression, localization, and function in metastatic CaP have not been investigated. Analysis of metadata and cell line models found increased DDX3 expression in metastatic versus primary CaP and benign prostate. Quantification of DDX3 expression in 320 human prostate samples, representing different stages of CaP progression, revealed an increase in epithelial whole cell, cytoplasmic, and nuclear DDX3 in primary CaP compared with benign prostate. In metastatic tissues, cytoplasmic DDX3 remained highly expressed, whereas nuclear DDX3 significantly decreased compared with primary CaP, suggesting a potential role for cytoplasmic DDX3 in metastatic CaP. Genetic and pharmacologic loss of function for DDX3 in metastatic CaP produced a significant decrease in cell viability, proliferation, and motility but did not affect apoptosis. The data suggest that cytoplasmic DDX3 is highly expressed in metastatic CaP and that inhibition of DDX3 affects metastatic growth by decreasing proliferation and motility. These findings introduce a novel role for cytoplasmic DDX3 in CaP progression and provide a foundation for clinically targeting DDX3 in metastatic CaP.

Inflammation is involved in many prostate pathologies including infection, benign prostatic hyperplasia, and prostate cancer. Preclinical models are critical to our understanding of disease mechanisms, yet few models are genetically tractable. Here, we present a comparative quantitative proteomic analysis of urine from mice with and without prostate-specific inflammation induced by conditional prostate epithelial IL-1β expression. Relative quantification and sample multiplexing was achieved using custom 4-plex N, N-dimethyl leucine (DiLeu) isobaric tags and nanoflow ultrahigh-performance liquid chromatography coupled to high-resolution tandem mass spectrometry. Each set of 4-plex DiLeu reagents allows four urine samples to be analyzed simultaneously, providing high-throughput and accurate quantification of urinary proteins. Proteins involved in the acute phase response, including haptoglobin, inter-α-trypsin inhibitor, and α1-antitrypsin 1-1, were differentially represented in the urine of mice with prostate inflammation. Mass spectrometry-based quantitative urinary proteomics represents a promising bioanalytical strategy for biomarker discovery and the elucidation of molecular mechanisms in urological research.

Prostate disease incidence, both benign and malignant, directly correlates with age. Men under 40 years of age are rarely diagnosed with benign or malignant prostate disease, while 90% of men over the age of 80 have histological evidence of benign disease (benign prostatic hyperplasia; BPH). Although rodent models have been invaluable in the study of disease progression and treatment efficacy, the effect of age is often not considered. In examining aged (24-month-old) mice, we observed changes within the lower urinary tract that is typically associated with lower urinary tract dysfunction (LUTD) similar to models of BPH. In this study, we identify LUTD using functional testing as well as various imaging technologies. We also characterize the histological differences within the lower urinary tract between young (2-month-old) and aged mice including proliferation, stromal remodeling, and collagen deposition. Additionally, we examined serum steroid hormone levels, as steroid changes drive LUTD in mice and are known to change with age. We conclude that, with age, changes in prostate function, consistent with LUTD, are a consequence. Therapeutic targeting of endocrine and prostatic factors including smooth muscle function, prostate growth and fibrosis are likely to reestablish normal urinary function.

Detailed mechanisms involved in prostate cancer (CaP) development and progression are not well understood. Current experimental models used to study CaP are not well suited to address this issue. Previously, we have described the hormonal progression of non-tumorigenic human prostate epithelial cells (BPH1) into malignant cells via tissue recombination. Here, we describe a method to derive human cell lines from distinct stages of CaP that parallel cellular, genetic and epigenetic changes found in patients with cancers. This BPH1-derived Cancer Progression (BCaP) model represents different stages of cancer. Using diverse analytical strategies, we show that the BCaP model reproduces molecular characteristics of CaP in human patients. Furthermore, we demonstrate that BCaP cells have altered gene expression of shared pathways with human and transgenic mouse CaP data, as well as, increasing genomic instability with TMPRSS2–ERG fusion in advanced tumor cells. Together, these cell lines represent a unique model of human CaP progression providing a novel tool that will allow the discovery and experimental validation of mechanisms regulating human CaP development and progression. This BPH1-derived Cancer Progression (BCaP) model represents different stages of cancer. The BCaP model reproduces molecular characteristics of prostate cancer. The cells have altered gene expression with TMPRSS2-ERG fusion representing a unique model for prostate cancer progression.

Objectives To describe the trend in the impact of lower urinary tract symptoms attributed to benign prostatic hyperplasia (LUTS/BPH) on a global scale using the Global Burden of Disease (GBD) database. Materials and methods Using the GBD database, worldwide data aggregated from registries and health systems from 1990 to 2017 were filtered for LUTS/BPH diagnoses. Calculation of years lived with disability (YLD) were compared with other urological diseases. YLD were calculated by a standardized method using assigned disability weights. The GBD-defined sociodemographic index (SDI) was used to assess impact of LUTS/BPH by global SDI quintile. Results Global Burden of Disease data over the 1990-2017 study period were summarized and global numbers and trends noted with other urological diseases for comparison. A total of 2 427 334 YLD were attributed to BPH in 2017 alone, almost three times more than those attributed to the next highest urological disease, prostate cancer (843 227 YLD). When stratified by SDI quintile, a much lower impact of BPH was found in the bottom three quintiles, despite this subset representing 66.9% of the 2017 world population. Conclusions Lower urinary tract symptoms attributed to benign prostatic hyperplasia exert a rapidly rising human burden far exceeding other urological diseases. As the population ages and men in a lower SDI enjoy increased life expectancy and decreased competing mortalities, a continually accelerating wave of LUTS/BPH can be forecast. These epidemiological trends have serious implications for the future allocation of resources and the global urological workforce.

Differentiating between indolent and aggressive prostate cancers (CaP) is important to decrease overtreatment and increase survival for men with the aggressive disease. Nucleolar prominence is a histologic hallmark of CaP; however, the expression, localization, and functional significance of specific nucleolar proteins have not been investigated thoroughly. The nucleolar protein block of proliferation 1 (BOP1) is associated with multiple cancers but has not been implicated in CaP thus far. Meta-analysis of publicly available data showed increased BOP1 expression in metastatic CaP and recurrent CaP, and was inversely associated with overall survival. Multiplexed immunohistochemistry was used to analyze expression and localization of BOP1 and nucleolar protein 56 in human tissue samples from various stages of CaP progression. Here, increased BOP1 expression was observed at later stages of CaP progression, coinciding with a localization change from nuclear to cytoplasmic. In patient samples, cytoplasmic BOP1 was also inversely associated with overall survival. In models of prostate cancer progression, BOP1 expression showed expression and localization similar to that in human patient samples. The functional significance of BOP1 in metastatic CaP was assessed by genetic knockdown, where BOP1 knockdown resulted in decreased proliferation and motility compared with control. Taken together, these data suggest prognostic significance of BOP1 expression and localization in CaP progression and provide a foundation for further investigation into the functional role of nucleolar proteins in advanced CaP.

Background/objectives To estimate associations between lower urinary tract symptoms (LUTS) and phenotypic frailty in older men. Design Cross-sectional study. Setting Community-dwelling men recruited from 2000 to 2002 from six U.S. academic centers for the Osteoporotic Fractures in Men Study. Participants A total of 5,979 men aged 65 and older. Measurements The independent variable was LUTS severity (none/mild, moderate, or severe) assessed with the American Urologic Association Symptom Index. Participants were categorized as frail, intermediate stage, or robust using an adapted Cardiovascular Health Study index (components: low lean mass, weakness, exhaustion, slowness, and low physical activity). Associations were estimated with odds ratios and 95% confidence intervals (CIs) from multivariable multinomial logistic regression models adjusted for potential confounders of age, other demographics, health-related behaviors, and comorbidities. Results The prevalence of frailty was 7%, 11%, and 18% among men with none/mild, moderate, and severe LUTS, respectively. Moderate and severe LUTS, overall and by storage and voiding subscores, were associated with higher odds of both intermediate stage and frailty in all models. After adjustment for confounders, the odds of frailty was 1.41 times higher among men with moderate LUTS (95% CI = 1.14-1.74) and 2.51 times higher among men with severe LUTS (95% CI = 1.76-3.55), compared with none/mild LUTS. Severe LUTS was associated with a greater odds of individual frailty components exhaustion and low physical activity. Conclusion The prevalence of phenotypic frailty is higher among older community-dwelling men with moderate or severe LUTS compared with those with mild or no LUTS. The positive association between LUTS severity and frailty among older men appears independent of age and known frailty risk factors. Although the temporal direction of this association and the utility of LUTS or frailty interventions in this population remain unclear, the high co-occurrence of these conditions could lead to earlier identification of frailty when clinically appropriate.

Introduction and Objective: Racial disparity in prostate cancer has been well established, with African American (AA) men having higher rates of diagnoses and death from the disease compared to Caucasian American (CA) men. AA men also have a high incidence of benign prostatic hyperplasia (BPH), a disease associated with lower urinary tract symptoms (LUTS) that affect >210 million men worldwide. Furthermore, AA men with BPH have an increased incidence of non-surgical treatment failure, larger prostates at time of surgery, and surgery occurring at a younger age. The use of selective estrogen receptor modulators (SERMs) in the treatment of BPH has been proposed, as an increase in ERα has been associated with disease progression. AA men have higher levels of circulating estrogens as compared to CA leading to an increased prenatal exposure to estrogens. Estrogen exposure has been shown to alter the epigenetic landscape of genes, and this prenatal exposure to estrogens could sensitize the AA men to altered steroid homeostasis leading to an increase susceptibility to BPH and an altered response to treatment. In this study, we examine the prostate expression and localization changes in estrogen receptors (ERα, ERβ) as well as steroid metabolism genes in AA and CA with or without BPH. Methods: To examine the impact of race on BPH, we examined prostate tissue from 66 men. We utilized 21 normal transition zone controls from radical prostatectomies, 8 normal transition zone controls from organ donors, and 37 BPH samples divided between CA and AA men. Using multispectral quantitative multiplex IHC, we examined the steroid hormone related protein expression of ERα, ERβ, CYP7B1, and AKR1C1 on each FFPE tissue section. We quantified the optical density of each protein of interest as well as examined colocalization and coexpression through cell and tissue segmentation. Results: In CA men, there is a dysregulation of ERα:ERβ homeostasis with BPH relative to normal as an increase in ERα and a decrease in ERβ expression was observed. Furthermore, an increase in CYP7B1, an enzyme that degrades ERβ ligands, was also observed. In AA men, we observed no difference between normal and BPH states, however in both normal and BPH prostate tissues, ERα and ERβ were increased relative to CA men. In addition, there is a decrease in AKR1C1, the enzyme that metabolizes DHT to an ERβ ligand. Conclusions: Our study supports the concept that differences in hormone pathways exist between AA and CA men. Understanding how these racial difference in steroid metabolism enzymes as well as ERs between CA and AA men with BPH could enhance treatment strategies for men with BPH.

Male lower urinary tract symptoms (LUTS) comprise a common syndrome of aging that negatively impacts quality of life. The etiology of LUTS is multifactorial, involving benign prostatic hyperplasia, smooth muscle and neurologic dysfunction, inflammation, sexually transmitted infections, fibrosis, and potentially dysbiosis, but this aspect remains poorly explored. We investigated whether the presence of infectious agents in urine might be associated with LUTS by combining next-generation DNA sequencing for virus discovery, microbiome analysis for characterization of bacterial communities, and mass spectrometry-based metabolomics. In urine from 29 LUTS cases and 9 controls from Wisconsin, we found a statistically significant association between a diagnosis of LUTS and the presence of JC virus (JCV), a common neurotropic human polyomavirus (Polyomaviridae,Betapolyomavirus) linked to severe neurologic disease in rare cases. This association (based on metagenomics) was not borne out when specific polymerase chain reaction (PCR) testing was applied to this set of samples, likely due to the greater sensitivity of PCR. Interestingly, urine metabolomics analysis identified dysregulation of metabolites associated with key LUTS processes. Microbiome analysis found no evidence of microbial community dysbiosis in LUTS cases, but JCV-positive samples contained moreAnaerococcusspecies, which are involved in polymicrobial infections of the urinary tract. Neither age nor body mass index were significantly associated with the presence of urinary JCV—in the initial group or in an additional, regionally distinct group. These data provide preliminary support the hypothesis that viruses such as JCV may play a role in the development or progression of LUTS, together with other infectious agents and host metabolic responses.

INTRODUCTION AND OBJECTIVE:Racial disparity in prostate cancer has been well established, with African American (AA) men having higher rates of diagnoses and death from the disease compared to Cauc...

INTRODUCTION AND OBJECTIVE:Approximately 30% of men with lower urinary tract symptoms (LUTS) are resistant to medical therapies and progress to surgical intervention. The pathological changes produ...

Mouse urinary behavior is quantifiable and is used to pinpoint mechanisms of voiding dysfunction and evaluate potential human therapies. Approaches to evaluate mouse urinary function vary widely among laboratories, however, complicating cross-study comparisons. Here, we describe development and multi-institutional validation of a new tool for objective, consistent, and rapid analysis of mouse void spot assay (VSA) data. Void Whizzard is a freely available software plugin for FIJI (a distribution of ImageJ) that facilitates VSA image batch processing and data extraction. We describe its features, demonstrate them by evaluating how specific VSA method parameters influence voiding behavior, and establish Void Whizzard as an expedited method for VSA analysis. This study includes control and obese diabetic mice as models of urinary dysfunction to increase rigor and ensure relevance across distinct voiding patterns. In particular, we show that Void Whizzard is an effective tool for quantifying nonconcentric overlapping void spots, which commonly confound analyses. We also show that mouse genetics are consistently more influential than assay design parameters when it comes to VSA outcomes. None of the following procedural modifications to reduce overlapping spots masked these genetic-related differences: reduction of VSA testing duration, water access during the assay period, placement of a wire mesh cage bottom on top of or elevated over the filter paper, treatment of mesh with a hydrophobic spray, and size of wire mesh opening. The Void Whizzard software and rigorous validation of VSA methodological parameters described here advance the goal of standardizing mouse urinary phenotyping for comprehensive urinary phenome analyses.

BackgroundMale lower urinary tract symptoms (LUTS) occur in more than half of men above 50 years of age. LUTS were traditionally attributed to benign prostatic hyperplasia (BPH) and therefore the clinical terminology often use LUTS and BPH interchangeably. More recently, LUTS were also linked to fibrogenic and inflammatory processes. We tested whether osteopontin (OPN), a pro-inflammatory and pro-fibrotic molecule, is increased in symptomatic BPH. We also tested whether prostate epithelial and stromal cells secrete OPN in response to pro-inflammatory stimuli and identified downstream targets of OPN in prostate stromal cells.MethodsImmunohistochemistry was performed on prostate sections obtained from the transition zone (TZ) of patients who underwent surgery (Holmium laser enucleation of the prostate) to relieve LUTS i.e. surgical BPH (S-BPH) or patients who underwent radical prostatectomy to remove low-grade prostate cancer (incidental BPH, I-BPH). Images of stained tissue sections were captured with a Nuance Multispectral Imaging system and histoscore, as a measure of OPN staining intensity, was determined with inForm software. OPN protein abundance was determined by Western blot. The ability of prostate cells to secrete osteopontin in response to IL-1β and TGF-β1 was determined in stromal (BHPrS-1) and epithelial (NHPrE-1 and BHPrE-1) cells by ELISA. qPCR was used to measure gene expression changes in these cells in response to OPN.ResultsOPN immunostaining (p=0.0107) and protein levels were more abundant in S-BPH than I-BPH. Staining was distributed across all cell types with highest levels in epithelial cells. Multiple OPN protein variants were identified in immortalized prostate stromal and epithelial cells. TGF-β1 stimulated OPN secretion by NHPrE-1 cells and both IL-1β and TGF-β1 stimulated OPN secretion by BHPrS-1 cells. Interestingly, recombinant OPN increased the mRNA expression ofCXCL1,CXCL2,CXCL8,PTGS2andIL6in BHPrS-1, but not in epithelial cell lines.ConclusionsOPN is more abundant in prostates of men with S-BPH compared to men with I-BPH. OPN secretion is stimulated by pro-inflammatory cytokines, and OPN acts directly on stromal cells to drive the synthesis of pro-inflammatory mRNAs. Pharmacological manipulation of prostatic OPN may have the potential to reduce LUTS by inhibiting both inflammatory and fibrotic pathways.

The study of male and female reproductive tract development requires expertise in two separate disciplines, developmental biology and endocrinology. For ease of experimentation and economy, the mouse has been used extensively as a model for human development and pathogenesis, and for the most part similarities in developmental processes and hormone action provide ample justification for the relevance of mouse models for human reproductive tract development. Indeed, there are many examples describing the phenotype of human genetic disorders that have a reasonably comparable phenotype in mice, attesting to the congruence between mouse and human development. However, anatomic, developmental and endocrinologic differences exist between mice and humans that (1) must be appreciated and (2) considered with caution when extrapolating information between all animal models and humans. It is critical that the investigator be aware of both the similarities and differences in organogenesis and hormone action within male and female reproductive tracts so as to focus on those features of mouse models with clear relevance to human development/pathology. This review, written by a team with extensive expertise in the anatomy, developmental biology and endocrinology of both mouse and human urogenital tracts, focusses upon the significant human/mouse differences, and when appropriate voices a cautionary note regarding extrapolation of mouse models for understanding development of human male and female reproductive tracts.

Aging is the single largest risk factor for many common diseases that burden public health. This is especially true in the prostate; as men age, the prostate undergoes a prototypical aging change, fibrosis. The aged fibrotic prostate causes urinary symptoms which will afflict nearly every man if they live long enough. However, the molecular mechanisms responsible for the aging-dependent promotion of fibrosis are largely unknown and understudied. In this study, we sought to reveal the contribution of mitochondrial dysfunction to fibrosis in the aging prostate, ultimately leading to prostate dysfunction, urinary symptoms, and overall poor health. The demise of mitochondrial function is well established in other aging-associated diseases but has not been investigated in the prostate. Our analysis revealed an increase in cellular senescence and mitochondrial dysfunction in BPH patient tissue compared to normal prostates. Furthermore, selective inhibition of mitochondrial complex I by rotenone in cultured human prostate fibroblasts led to myofibroblast phenoconversion as characterized by increased expression of Col1a1, Col3a1, and ACTA2. To determine the contribution of mitochondrial dysfunction on fibrosis and lower urinary tract dysfunction (LUTD), we examined the prostate lobes in both aging and steroid-induced LUTD in mice. These models have been extensively characterized and exhibit an age-mediated increase in LUTD and fibrosis. We observed a decrease in mitochondrial function and an increase in cellular senescence corresponding to an increase in fibrosis and LUTD. This suggests that normal aging-dependent reductions in mitochondrial complex I function in the prostate may promote fibrosis and contribute to urinary dysfunction.

Despite the advancement of transgenic and gene knockout animal models in the prostate cancer research, there is still a need for utilizing xenograft models. Xenografts can be grown in multiple sites/organs within immunocompromised animals such as mice and rats. Although prostate xenografts have been derived from many species, human cells and tissues are the most commonly used due to their potential clinical significance. Xenograft models that progress from one state or stage to another are commonly used to address important scientific questions including malignant transformation, metastatic spread, and castration resistance. Utilization of xenografts are commonly being used to assess the biology and genetics of prostate cancer, as well as, for therapeutic benefit. In addition to models for the study of prostate cancer, xenografts are also utilized as a tool in precision medicine where patient derived xenografts (PDX) can be grown in multiple animals and assessed for therapeutic efficacy. The popularity of such xenograft models and PDXs have led to availability of these resources through public and commercial institutions. In this review, we describe both traditional and emerging models of prostate cancer and their potential uses. Further development of current models and introduction of new models will likely provide new insights and better understanding of prostatic carcinogenesis and progression.