Androgen receptor splice variants drive castration-resistant prostate cancer metastasis by activating distinct transcriptional programs.

One critical mechanism through which prostate cancer (PCa) adapts to treatments targeting androgen receptor (AR) signaling is the emergence of ligand-binding domain-truncated and constitutively active AR splice variants, particularly AR-V7. While AR-V7 has been intensively studied, its ability to activate distinct biological functions compared to the full-length AR (AR-FL), and its role in regulating the metastatic progression of castration-resistant PCa (CRPC), remains unclear. Our study found that, under castrated conditions, AR-V7 strongly induced osteoblastic bone lesions, a response not observed with AR-FL overexpression. Through combined ChIP-seq, ATAC-seq, and RNA-seq analyses, we demonstrated that AR-V7 uniquely accesses the androgen-responsive elements in compact chromatin regions, activating a distinct transcription program. This program was highly enriched for genes involved in epithelial-mesenchymal transition and metastasis. Notably, we discovered that SOX9, a critical metastasis driver gene, was a direct target and downstream effector of AR-V7. Its protein expression was dramatically upregulated in AR-V7-induced bone lesions. Moreover, we found that Ser81 phosphorylation enhanced AR-V7's pro-metastasis function by selectively altering its specific transcription program. Blocking this phosphorylation with CDK9 inhibitors impaired the AR-V7-mediated metastasis program. Overall, our study has provided molecular insights into the role of AR splice variants in driving the metastatic progression of CRPC.

The use of beta-sitosterol for the treatment of prostate cancer and benign prostatic hyperplasia.

Herbal supplements are widely used to enhance prostate health. These supplements may contain several types of plant sterols, vitamins, and minerals. By weight, however, plant sterols make up an abundant ingredient component, with saw palmetto extract or its primary component, beta-sitosterol, often comprising the most abundant sterol. Saw palmetto extract/beta-sitosterol has been shown to promote anti-tumorigenic processes in prostate cancer cells and rodent models of prostate cancer. It has also been shown to inhibit the 5α-reductase enzyme, thereby behaving similarly to finasteride and dutasteride, which are widely used to treat prostatic enlargement, or benign prostatic hyperplasia (BPH). The aim of this study is to critically examine in vitro, in vivo, and human clinical studies to assess the safety and clinical utility of herbal supplements containing saw palmetto extract/beta-sitosterol for prostate health. The results of this study suggest multiple mechanisms through which beta-sitosterol represses prostate cancer in vitro and in vivo, particularly through its pro-apoptotic effect on prostate epithelial cells. Multiple studies also show that beta-sitosterol significantly improves lower urinary tract symptoms (LUTS) associated with BPH, but to an extent that is generally less effective than that achieved by pharmaceutical grade alpha-adrenergic receptor antagonists or 5α-reductase inhibitors. This latter finding suggests that supplements containing beta-sitosterol might be most appropriate for younger men with minimal LUTS who don't wish to embark on a clinical drug regimen for BPH treatment.

Demethylation of EHMT1/GLP Protein Reprograms Its Transcriptional Activity and Promotes Prostate Cancer Progression.

Epigenetic reprogramming, mediated by genomic alterations and dysregulation of histone reader and writer proteins, plays a critical role in driving prostate cancer progression and treatment resistance. However, the specific function and regulation of EHMT1 (also known as GLP) and EHMT2 (also known as G9A), well-known histone 3 lysine 9 methyltransferases, in prostate cancer progression remain poorly understood. Through comprehensive investigations, we discovered that both EHMT1 and EHMT2 proteins have the ability to activate oncogenic transcription programs in prostate cancer cells. Silencing EHMT1/2 or targeting their enzymatic activity with small-molecule inhibitors can markedly decrease prostate cancer cell proliferation and metastasis in vitro and in vivo. In-depth analysis of posttranslational modifications of EHMT1 protein revealed the presence of methylation at lysine 450 and 451 residues in multiple prostate cancer models. Notably, we found that lysine 450 can be demethylated by LSD1. Strikingly, concurrent demethylation of both lysine residues resulted in a rapid and profound expansion of EHMT1's chromatin binding capacity, enabling EHMT1 to reprogram the transcription networks in prostate cancer cells and activate oncogenic signaling pathways. Overall, our studies provide valuable molecular insights into the activity and function of EHMT proteins during prostate cancer progression. Moreover, we propose that the dual-lysine demethylation of EHMT1 acts as a critical molecular switch, triggering the induction of oncogenic transcriptional reprogramming in prostate cancer cells. These findings highlight the potential of targeting EHMT1/2 and their demethylation processes as promising therapeutic strategies for combating prostate cancer progression and overcoming treatment resistance. Significance: In this study, we demonstrate that EHMT1 and EHMT2 proteins drive prostate cancer development by transcriptionally activating multiple oncogenic pathways. Mechanistically, the chromatin binding of EHMT1 is significantly expanded through demethylation of both lysine 450 and 451 residues, which can serve as a critical molecular switch to induce oncogenic transcriptional reprogramming in prostate cancer cells.

SETD7 functions as a transcription repressor in prostate cancer via methylating FOXA1.

Dysregulation of histone lysine methyltransferases and demethylases is one of the major mechanisms driving the epigenetic reprogramming of transcriptional networks in castration-resistant prostate cancer (CRPC). In addition to their canonical histone targets, some of these factors can modify critical transcription factors, further impacting oncogenic transcription programs. Our recent report demonstrated that LSD1 can demethylate the lysine 270 of FOXA1 in prostate cancer (PCa) cells, leading to the stabilization of FOXA1 chromatin binding. This process enhances the activities of the androgen receptor and other transcription factors that rely on FOXA1 as a pioneer factor. However, the identity of the methyltransferase responsible for FOXA1 methylation and negative regulation of the FOXA1-LSD1 oncogenic axis remains unknown. SETD7 was initially identified as a transcriptional activator through its methylation of histone 3 lysine 4, but its function as a methyltransferase on nonhistone substrates remains poorly understood, particularly in the context of PCa progression. In this study, we reveal that SETD7 primarily acts as a transcriptional repressor in CRPC cells by functioning as the major methyltransferase targeting FOXA1-K270. This methylation disrupts FOXA1-mediated transcription. Consistent with its molecular function, we found that SETD7 confers tumor suppressor activity in PCa cells. Moreover, loss of SETD7 expression is significantly associated with PCa progression and tumor aggressiveness. Overall, our study provides mechanistic insights into the tumor-suppressive and transcriptional repression activities of SETD7 in mediating PCa progression and therapy resistance.

LSD1 Inhibition Disrupts Super-Enhancer-Driven Oncogenic Transcriptional Programs in Castration-Resistant Prostate Cancer.

The lysine demethylase LSD1 (also called KDM1A) plays important roles in promoting multiple malignancies including both hematologic cancers and solid tumors. LSD1 targets histone and nonhistone proteins and can function as a transcriptional corepressor or coactivator. LSD1 has been reported to act as a coactivator of androgen receptor (AR) in prostate cancer and to regulate the AR cistrome via demethylation of its pioneer factor FOXA1. A deeper understanding of the key oncogenic programs targeted by LSD1 could help stratify prostate cancer patients for treatment with LSD1 inhibitors, which are currently under clinical investigation. In this study, we performed transcriptomic profiling in an array of castration-resistant prostate cancer (CRPC) xenograft models that are sensitive to LSD1 inhibitor treatment. Impaired tumor growth by LSD1 inhibition was attributed to significantly decreased MYC signaling, and MYC was found to be a consistent target of LSD1. Moreover, LSD1 formed a network with BRD4 and FOXA1 and was enriched at super-enhancer regions exhibiting liquid-liquid phase separation. Combining LSD1 inhibitors with BET inhibitors exhibited strong synergy in disrupting the activities of multiple drivers in CRPC, thereby inducing significant growth repression of tumors. Importantly, the combination treatment showed superior effects than either inhibitor alone in disrupting a subset of newly identified CRPC-specific super-enhancers. These results provide mechanistic and therapeutic insights for cotargeting two key epigenetic factors and could be rapidly translated in the clinic for CRPC patients. SIGNIFICANCE: LSD1 drives prostate cancer progression by activating super-enhancer-mediated oncogenic programs, which can be targeted with the combination of LSD1 and BRD4 inhibitors to suppress the growth of CRPC.

Increased AR expression in castration-resistant prostate cancer rapidly induces AR signaling reprogramming with the collaboration of EZH2.

Elevated androgen receptor (AR) expression is a hallmark of castration-resistant prostate cancer (CRPC) and contributes to the restoration of AR signaling under the conditions of androgen deprivation. However, whether overexpressed AR alone with the stimulation of castrate levels of androgens can be sufficient to induce the reprogramming of AR signaling for the adaptation of prostate cancer (PCa) cells remains unclear. In this study, we used a PCa model with inducible overexpression of AR to examine the acute effects of AR overexpression on its cistrome and transcriptome. Our results show that overexpression of AR alone in conjunction with lower androgen levels can rapidly redistribute AR chromatin binding and activates a distinct transcription program that is enriched for DNA damage repair pathways. Moreover, using a recently developed bioinformatic tool, we predicted the involvement of EZH2 in this AR reprogramming and subsequently identified a subset of AR/EZH2 co-targeting genes, which are overexpressed in CRPC and associated with worse patient outcomes. Mechanistically, we found that AR-EZH2 interaction is impaired by the pre-castration level of androgens but can be recovered by the post-castration level of androgens. Overall, our study provides new molecular insights into AR signaling reprogramming with the engagement of specific epigenetic factors.

The IL-4/IL-13 signaling axis promotes prostatic fibrosis.

BACKGROUND: Lower urinary tract symptoms (LUTS) are a costly and pervasive medical problem for millions of aging men. Recent studies have showed that peri-urethral tissue fibrosis is an untreated pathobiology contributing to LUTS. Fibrosis results from excessive extracellular matrix deposition which increases transition zone and peri-urethral tissue stiffness and compromises prostatic urethral flexibility and compliance, producing urinary obstructive symptoms. Inflammatory cells, including neutrophils, macrophages, and T-lymphocytes, secrete a medley of pro-fibrotic proteins into the prostatic microenvironment, including IFNγ, TNFα, CXC-type chemokines, and interleukins, all of which have been implicated in inflammation-mediated fibrosis. Among these, IL-4 and IL-13 are of particular interest because they share a common signaling axis that, as shown here for the first time, promotes the expression and maintenance of IL-4, IL-13, their cognate receptors, and ECM components by prostate fibroblasts, even in the absence of immune cells. Based on studies presented here, we hypothesize that the IL-4/IL-13 axis promotes prostate fibroblast activation to ECM-secreting cells. METHODS: N1 or SFT1 immortalized prostate stromal fibroblasts were cultured and treated, short- or long-term, with pro-fibrotic proteins including IL-4, IL-13, TGF-ß, TNF-α, IFNγ, with or without prior pre-treatment with antagonists or inhibitors. Protein expression was assessed by immunohistochemistry, immunofluorescence, ELISA, immunoblot, or Sircoll assays. Transcript expression levels were determined by qRT-PCR. Intact cells were counted using WST assays. RESULTS: IL-4Rα, IL-13Rα1, and collagen are concurrently up-regulated in human peri-urethral prostate tissues from men with LUTS. IL-4 and IL-13 induce their own expression as well as that of their cognate receptors, IL-4Rα and IL-13Rα1. Low concentrations of IL-4 or IL-13 act as cytokines to promote prostate fibroblast proliferation, but higher (>40ng/ml) concentrations repress cellular proliferation. Both IL-4 and IL-13 robustly and specifically promote collagen transcript and protein expression by prostate stromal fibroblasts in a JAK/STAT-dependent manner. Moreover, IL-4 and IL-13-mediated JAK/STAT signaling is coupled to activation of the IL-4Rα receptor. CONCLUSIONS: Taken together, these studies show that IL-4 and IL-13 signal through the IL-4Rα receptor to activate JAK/STAT signaling, thereby promoting their own expression, that of their cognate receptors, and collagens. These finding suggest that the IL-4/IL-13 signaling axis is a powerful, but therapeutically targetable, pro-fibrotic mechanism in the lower urinary tract.

Exploiting the tumor-suppressive activity of the androgen receptor by CDK4/6 inhibition in castration-resistant prostate cancer.

The androgen receptor (AR) plays a pivotal role in driving prostate cancer (PCa) development. However, when stimulated by high levels of androgens, AR can also function as a tumor suppressor in PCa cells. While the high-dose testosterone (high-T) treatment is currently being tested in clinical trials of castration-resistant prostate cancer (CRPC), there is still a pressing need to fully understand the underlying mechanism and thus develop treatment strategies to exploit this tumor-suppressive activity of AR. In this study, we demonstrate that retinoblastoma (Rb) family proteins play a central role in maintaining the global chromatin binding and transcriptional repression program of AR and that Rb inactivation desensitizes CRPC to the high-dose testosterone treatment in vitro and in vivo. Using a series of patient-derived xenograft (PDX) CRPC models, we further show that the efficacy of high-T treatment can be fully exploited by a CDK4/6 inhibitor, which strengthens the chromatin binding of the Rb-E2F repressor complex by blocking the hyperphosphorylation of Rb proteins. Overall, our study provides strong mechanistic and preclinical evidence on further developing clinical trials to combine high-T with CDK4/6 inhibitors in treating CRPC.

RB1 loss in castration-resistant prostate cancer confers vulnerability to LSD1 inhibition.

Genomic loss of RB1 is a common alteration in castration-resistant prostate cancer (CRPC) and is associated with poor patient outcomes. RB1 loss is also a critical event that promotes the neuroendocrine transdifferentiation of prostate cancer (PCa) induced by the androgen receptor (AR) signaling inhibition (ARSi). The loss of Rb protein disrupts the Rb-E2F repressor complex and thus hyperactivates E2F transcription activators. While the impact of Rb inactivation on PCa progression and linage plasticity has been previously studied, there is a pressing need to fully understand underlying mechanisms and identify vulnerabilities that can be therapeutically targeted in Rb-deficient CRPC. Using an integrated cistromic and transcriptomic analysis, we have characterized Rb activities in multiple CRPC models by identifying Rb-directly regulated genes and revealed that Rb has distinct binding sites and targets in CRPC with different genomic backgrounds. Significantly, we show that E2F1 chromatin binding and transcription activity in Rb-deficient CRPC are highly dependent on LSD1/KDM1A, and that Rb inactivation sensitizes CRPC tumor to the LSD1 inhibitor treatment. These results provide new molecular insights into Rb activity in PCa progression and suggest that targeting LSD1 activity with small molecule inhibitors may be a potential treatment strategy to treat Rb-deficient CRPC.

PCNA-associated factor (KIAA0101/PCLAF) overexpression and gene copy number alterations in hepatocellular carcinoma tissues.

BACKGROUND: PCNA-associated factor, the protein encoded by the KIAA0101/PCLAF gene, is a cell-cycle regulated oncoprotein that regulates DNA synthesis, maintenance of DNA methylation, and DNA-damage bypass, through the interaction with the human sliding clamp PCNA. KIAA0101/PCLAF is overexpressed in various cancers, including hepatocellular carcinoma (HCC). However, it remains unknown whether KIAA0101/PCLAF overexpression is coupled to gene amplification in HCC. METHODS: KIAA0101/PCLAF mRNA expression levels were assessed by quantitative real-time PCR (qRT-PCR) in 40 pairs of snap-frozen HCC and matched-non-cancerous tissues. KIAA0101/PCLAF gene copy numbers were evaluated by droplet digital PCR (ddPCR) in 36 pairs of the tissues, and protein expression was detected by immunohistochemistry (IHC) in 81 pairs of formalin-fixed paraffin-embedded (FFPE) tissues. The KIAA0101/PCLAF gene copy number alteration and RNA expression was compared by Spearman correlation. The relationships between KIAA0101 protein expression and other clinicopathological parameters, including Ki-67, p53, and HBsAg protein expression in HCC tissues, were evaluated using Chi-square test. RESULTS: Our results demonstrated that KIAA0101/PCLAF mRNA levels were significantly higher in HCC than in the matched-non-cancerous tissues (p < 0.0001). The high KIAA0101/PCLAF mRNA levels in HCC were associated with poor patient survival. The KIAA0101/PCLAF gene was not amplified in HCC, and KIAA0101/PCLAF gene copy numbers were not associated with KIAA0101/PCLAF transcript levels. KIAA0101 protein was overexpressed in the majority of HCC tissues (77.8%) but was not detectable in matched-non-cancerous tissues. Significant correlations between the expression of KIAA0101 protein in HCC tissues and p53 tumor suppressor protein (p = 0.002) and Ki-67 proliferation marker protein (p = 0.017) were found. However, KIAA0101 protein levels in HCC tissues were not correlated with patient age, tumor size, serum AFP level, or the HBsAg expression. CONCLUSIONS: KIAA0101/PCLAF mRNA and protein overexpression is frequently observed in HCC but without concurrent KIAA0101/PCLAF gene amplification. Significant correlations between the expression of KIAA0101 protein and p53 and Ki-67 proteins were observed in this study. Thus, detection of KIAA0101/PCLAF mRNA/protein might be used, along with the detection of p53 and Ki-67 proteins, as potential biomarkers to select candidate patients for further studies of novel HCC treatment related to these targets.

Susceptibility-Associated Genetic Variation in NEDD9 Contributes to Prostate Cancer Initiation and Progression.

Although American men of European ancestry represent the largest population of patients with prostate cancer, men of African ancestry are disproportionately affected by prostate cancer, with higher prevalence and worse outcomes. These racial disparities in prostate cancer are due to multiple factors, but variations in genomic susceptibility such as SNP may play an important role in determining cancer aggressiveness and treatment outcome. Using public databases, we have identified a prostate cancer susceptibility SNP at an intronic enhancer of the neural precursor expressed, developmentally downregulated 9 (NEDD9) gene, which is strongly associated with increased risk of patients with African ancestry. This genetic variation increased expression of NEDD9 by modulating the chromatin binding of certain transcription factors, including ERG and NANOG. Moreover, NEDD9 displayed oncogenic activity in prostate cancer cells, promoting prostate cancer tumor growth and metastasis in vitro and in vivo. Together, our study provides novel insights into the genetic mechanisms driving prostate cancer racial disparities. SIGNIFICANCE: A prostate cancer susceptibility genetic variation in NEDD9, which is strongly associated with the increased risk of patients with African ancestry, increases NEDD9 expression and promotes initiation and progression of prostate cancer.See related commentary by Mavura and Huang, p. 3764.

Chromatin binding of FOXA1 is promoted by LSD1-mediated demethylation in prostate cancer.

FOXA1 functions as a pioneer transcription factor by facilitating the access to chromatin for steroid hormone receptors, such as androgen receptor and estrogen receptor1-4, but mechanisms regulating its binding to chromatin remain elusive. LSD1 (KDM1A) acts as a transcriptional repressor by demethylating mono/dimethylated histone H3 lysine 4 (H3K4me1/2)5,6, but also acts as a steroid hormone receptor coactivator through mechanisms that are unclear. Here we show, in prostate cancer cells, that LSD1 associates with FOXA1 and active enhancer markers, and that LSD1 inhibition globally disrupts FOXA1 chromatin binding. Mechanistically, we demonstrate that LSD1 positively regulates FOXA1 binding by demethylating lysine 270, adjacent to the wing2 region of the FOXA1 DNA-binding domain. Acting through FOXA1, LSD1 inhibition broadly disrupted androgen-receptor binding and its transcriptional output, and dramatically decreased prostate cancer growth alone and in synergy with androgen-receptor antagonist treatment in vivo. These mechanistic insights suggest new therapeutic strategies in steroid-driven cancers.

Impact of sex, androgens, and prostate size on C57BL/6J mouse urinary physiology: urethral histology.

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.

Insight and Resources From a Study of the "Impact of Sex, Androgens, and Prostate Size on C57BL/6J Mouse Urinary Physiology.

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.

Ultrasonography of the Adult Male Urinary Tract for Urinary Functional Testing.

The incidence of clinical benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS) is increasing due to the aging population, resulting in a significant economic and quality of life burden. Transgenic and other mouse models have been developed to recreate various aspects of this multifactorial disease; however, methods to accurately quantitate urinary dysfunction and the effectiveness of new therapeutic options are lacking. Here, we describe a method that can be used to measure bladder volume and detrusor wall thickness, urinary velocity, void volume and void duration, and urethral diameter. This would allow for the evaluation of disease progression and treatment efficacy over time. Mice were anesthetized with isoflurane, and the bladder was visualized by ultrasound. For non-contrast imaging, a 3D image was taken of the bladder to calculate volume and evaluate shape; the bladder wall thickness was measured from this image. For contrast-enhanced imaging, a catheter was placed through the dome of the bladder using a 27-gauge needle connected to a syringe by PE50 tubing. A bolus of 0.5 mL of contrast was infused into the bladder until a urination event occurred. Urethral diameter was determined at the point of the Doppler velocity sample window during the first voiding event. Velocity was measured for each subsequent event yielding a flow rate. In conclusion, high frequency ultrasound proved to be an effective method for assessing bladder and urethral measurements during urinary function in mice. This technique may be useful in the assessment of novel therapies for BPH/LUTS in an experimental setting.

Impact of sex, androgens, and prostate size on C57BL/6J mouse urinary physiology: functional assessment.

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.

Void sorcerer: an open source, open access framework for mouse uroflowmetry.

Observational and experimental studies of rodent voiding behaviors have greatly contributed to our understanding of lower urinary tract function including the complex social, environmental, and internal stimuli that affect voiding in health and models of disease. Void spot assays (VSA), cystometry (awake or anesthetized), and uroflowmetry are techniques commonly used in rodent models to assess voiding. Uroflowmetry is non-invasive and can be performed multiple times in the same freely moving animals and can be used to generate synchronized video corresponding to each void to characterize micturition patterns (e.g., droplets versus solid stream). However, approaches to evaluate uroflowmetry in rodent models vary widely across laboratories. Most importantly, an open access software to run these tests is not freely available (although complete systems are commercially available), limiting use of this important assay. We developed the Void Sorcerer, an uroflowmetry system for mice for reliable determination of frequency, voided volume, voiding duration, interval times between micturitions, and flow rate. This report provides a detailed description of how to build this system and includes open access software for developing uroflowmetry capability in their laboratories and improve upon it in a cost-effective manner. Our goals are to improve access, increase reproducibility among laboratories, and facilitate standardizing testing procedures.

Prostate Transition Zone Fibrosis is Associated with Clinical Progression in the MTOPS Study.

PURPOSE: Medications targeting androgen receptor activity (eg finasteride) or smooth muscle contractility (eg doxazosin) do not resolve lower urinary tract symptoms indicative of lower urinary tract dysfunction in an important subgroup of men. Recently fibrosis has been implicated as another pathobiology contributing to male lower urinary tract symptoms but to our knowledge no systematic studies have been done to assess fibrosis in the context of medical treatment. We determine whether fibrotic changes in the prostate transition zone are associated with an increased risk of clinical progression in participants treated with doxazosin, finasteride or finasteride plus doxazosin in the MTOPS (Medical Therapy of Prostatic Symptoms) study. MATERIALS AND METHODS: Transition zone biopsy tissues from men who did or did not experience clinical progression on placebo, doxazosin, finasteride or combination therapy were assessed for collagen content and architectural changes using picrosirius red birefringence and CT-FIRE (Curvelet Transform-Fiber Extraction) analysis. Correlations were made with annotated demographic and clinical data. Statistical analyses were done with the Pearson correlation coefficient, ANOVA and the t-test. RESULTS: High levels of wavy, aligned prostate transition zone collagen significantly correlated with an increased risk of clinical progression among MTOPS trial participants treated with doxazosin plus finasteride, particularly those with a high body mass index. CONCLUSIONS: Fibrotic changes in the prostate transition zone are associated with an increased risk of clinical progression in men treated with doxazosin plus finasteride. Antifibrotic therapeutics might provide a new treatment approach in men with lower urinary tract dysfunction who do not respond to current medical treatment approaches.

TMPRSS2-ERG activates NO-cGMP signaling in prostate cancer cells.

The aberrant activation of the ERG oncogenic pathway due to the TMPRSS2-ERG gene fusion is the major event that contributes to prostate cancer (PCa) development. However, the critical downstream effectors that can be therapeutically targeted remain to be identified. In this study, we have found that the expression of the α1 and ß1 subunits of soluble guanylyl cyclase (sGC) was directly and specifically regulated by ERG in vitro and in vivo and was significantly associated with TMPRSS2-ERG fusion in clinical PCa cohorts. sGC is the major mediator of nitric oxide (NO)-cGMP signaling in cells that, upon NO binding, catalyzes the synthesis of cGMP and subsequently activates protein kinase G (PKG). We showed that cGMP synthesis was significantly elevated by ERG in PCa cells, leading to increased PKG activity and cell proliferation. Importantly, we also demonstrated that sGC inhibitor treatment repressed tumor growth in TMPRSS2-ERG-positive PCa xenograft models and can act in synergy with a potent AR antagonist, enzalutamide. This study strongly suggests that targeting NO-cGMP signaling pathways may be a novel therapeutic strategy to treat PCa with TMPRSS2-ERG gene fusion.