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FOXA family proteins act as pioneer factors by remodeling compact chromatin structures. FOXA1 is crucial for the chromatin binding of the androgen receptor (AR) in both normal prostate epithelial cells and the luminal subtype of prostate cancer (PCa). Recent studies have highlighted the emergence of FOXA2 as an adaptive response to AR signaling inhibition treatments. However, the role of the FOXA1 to FOXA2 transition in regulating cancer lineage plasticity remains unclear. Our study demonstrates that FOXA2 binds to distinct classes of developmental enhancers in multiple AR-independent PCa subtypes, with its binding depending on LSD1. Moreover, we reveal that FOXA2 collaborates with JUN at chromatin and promotes transcriptional reprogramming of AP-1 in lineage-plastic cancer cells, thereby facilitating cell state transitions to multiple lineages. Overall, our findings underscore the pivotal role of FOXA2 as a pan-plasticity driver that rewires AP-1 to induce the differential transcriptional reprogramming necessary for cancer cell lineage plasticity., (© 2024. The Author(s).)

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 with the full-length AR (AR-FL), and its role in regulating the metastatic progression of castration-resistant PCa (CRPC), remain 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.

Herbal supplements containing several types of plant sterols, vitamins, and minerals, are marketed for prostate health. In the majority of these supplements, the most abundant plant sterol is saw palmetto extract or its' principal component, beta-sitosterol. In terms of prostate health, previous work almost exclusively focused on the effects of beta-sitosterol on prostatic epithelium, with little attention paid to the effects on prostatic stroma. This omission is a concern, as the abnormal accumulation of collagen, or fibrosis, of the prostatic stroma has been identified as a factor contributing to lower urinary tract symptoms and dysfunction in aging men. To address whether beta-sitosterol may be promoting prostatic fibrosis, immortalized and primary prostate stromal fibroblasts were subjected to immunoblotting, immunofluorescence, qRT-PCR, ELISA, and image quantitation and analysis techniques to elucidate the effects of beta-sitosterol on cell viability and collagen expression and cellular localization. The results of these studies show that beta-sitosterol is nontoxic to prostatic fibroblasts and does not stimulate collagen production by these cells. However, beta-sitosterol alters collagen distribution and sequesters collagen within prostatic fibroblasts, likely in an age-dependent manner. This is a significant finding as prostate health supplements are used predominantly by middle aged and older men who may, then, be affected disproportionately by these effects.

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., Competing Interests: None., (AJCEU Copyright © 2023.)

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., (© 2023 The Authors; Published by the American Association for Cancer Research.)

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.

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., (©2023 American Association for Cancer Research.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Labaf, Li, Ting, Karno, Zhang, Gao, Patalano, Macoska, Zarringhalam, Han and Cai.)

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-β, 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., Competing Interests: The authors have declared that no competing interests exist.

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., Competing Interests: Declaration of interests No potential conflicts of interest were disclosed., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

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 ., (©2021 American Association for Cancer Research.)

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.

FOXA1 functions as a pioneer transcription factor by facilitating the access to chromatin for steroid hormone receptors, such as androgen receptor and estrogen receptor 1-4 , 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.

Inference of active regulatory mechanisms underlying specific molecular and environmental perturbations is essential for understanding cellular response. The success of inference algorithms relies on the quality and coverage of the underlying network of regulator-gene interactions. Several commercial platforms provide large and manually curated regulatory networks and functionality to perform inference on these networks. Adaptation of such platforms for open-source academic applications has been hindered by the lack of availability of accurate, high-coverage networks of regulatory interactions and integration of efficient causal inference algorithms. In this work, we present CIE, an integrated platform for causal inference of active regulatory mechanisms form differential gene expression data. Using a regularized Gaussian Graphical Model, we construct a transcriptional regulatory network by integrating publicly available ChIP-seq experiments with gene-expression data from tissue-specific RNA-seq experiments. Our GGM approach identifies high confidence transcription factor (TF)-gene interactions and annotates the interactions with information on mode of regulation (activation vs. repression). Benchmarks against manually curated databases of TF-gene interactions show that our method can accurately detect mode of regulation. We demonstrate the ability of our platform to identify active transcriptional regulators by using controlled in vitro overexpression and stem-cell differentiation studies and utilize our method to investigate transcriptional mechanisms of fibroblast phenotypic plasticity., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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.

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 Statement: The search for an ideal "off-the-shelf" biomaterial for augmentation cystoplasty remains elusive and current scaffold configurations are hampered by mechanical and biocompatibility restrictions. In addition, preclinical evaluations of potential scaffold designs for bladder repair are limited by the lack of tractable large animal models of obstructive bladder disease that can mimic clinical pathology. The results of this study describe a novel, minimally invasive, porcine model of partial bladder outlet obstruction that simulates clinically relevant phenotypes. Utilizing this model, we demonstrate that acellular, bi-layer silk fibroin grafts can support the formation of vascularized, innervated bladder tissues with functional properties.

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 β1 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.

Background: Several studies show that prostatic fibrosis is associated with male lower urinary tract dysfunction (LUTD). Development of fibrosis is typically attributed to signaling through the transforming growth factor β (TGF-β) pathway, but our laboratory has demonstrated that in vitro treatment of human prostatic fibroblasts with the C-X-C motif chemokine ligand 12 (CXCL12) chemokine stimulates myofibroblast phenoconversion and that CXCL12 has the capacity to activate profibrotic pathways in these cells in a TGF-β-independent manner. We have previously reported that feeding mice high-fat diet (HFD) results in obesity, type II diabetes, increased prostatic fibrosis, and urinary voiding dysfunction. The purpose of this study was to test the hypothesis that in vivo blockade of the CXCL12/CXCR4 axis would inhibit the development of fibrosis-mediated LUTD in HFD-fed mice., Methods: Two-month-old male senescence-accelerated mouse prone-6 mice were fed either a HFD or low-fat diet (LFD) for 8 months. Half of each dietary group were given constant access to normal water or water that contained the C-X-C chemokine receptor type 4 (CXCR4; CXCL12 receptor) antagonist CXCR4AIII. At the conclusion of the study, mice were weighed, subjected to oral glucose tolerance testing and cystometry, and lower urinary tract tissues collected and assessed for collagen content., Results: HFD-fed mice became significantly obese, insulin resistant, and hyperglycemic, consistent with acquisition of metabolic syndrome, compared with LFD-fed mice. Anesthetized cystometry demonstrated that HFD-fed mice experienced significantly longer intercontractile intervals and greater functional bladder capacity than LFD-fed mice. Immunohistochemistry demonstrated high levels of CXCR4 and CXCR7 staining in mouse prostate epithelial and stromal cells. Picrosirius red staining indicated significantly greater periurethral collagen deposition in the prostates of HFD than LFD-fed mice. Treatment with the CXCR4 antagonist CXCR4AIII did not affect acquisition of metabolic syndrome but did reduce both urinary voiding dysfunction and periurethral prostate collagen accumulation., Conclusions: This is the first study to report that obesity-induced lower urinary tract fibrosis and voiding dysfunction can be repressed by antagonizing the activity of the CXCR4 chemokine receptor in vivo. These data suggest that targeting the CXCL12/CXCR4 signaling pathway may be a clinical option for the prevention or treatment of human male LUTD., (© 2019 Wiley Periodicals, Inc.)

Background: Metabolic syndrome contributing to nonalcoholic fatty liver disease (NAFLD) can lead to hepatic dysfunction, steatohepatitis, cirrhosis, and hepatocellular carcinoma., Aims: In this study, we tested whether diet-induced fatty liver in a mouse model physiologically mimicked human NAFLD, and whether transcriptional alterations in mouse fatty liver signified risk for the development of hepatitis, cirrhosis, and/or hepatocellular carcinoma., Methods: SAMP6 strain mice were fed a low-fat diet or high-fat diet (HFD) for 6 months. Mouse livers were isolated and subjected to histology, immunohistochemistry, and whole transcriptome RNA sequencing. Sequences were aligned to the mouse reference genome, and gene expression signatures were analyzed using bioinformatics tools including Cufflinks, Pathview, Cytoscape, ClueGO, and GOstats., Results: Consistent with NAFLD, livers from HFD-fed mice demonstrated steatosis, high levels of inflammation, an up-regulation of genes encoding proteins associated with the complement pathway and immune responses, and down-regulation of those associated with metabolic processes. These livers also showed an up-regulation of genes associated with fibrosis and malignant transformation but no histological evidence of either pathobiology or DNA damage., Conclusions: HFD-fed mice exhibited NAFLD that had incompletely transitioned from fatty liver to NASH. Importantly, bioinformatics approaches identified pre-fibrotic and premalignant signatures, suggesting that the pathogenesis of both fibrosis and cancer may initiate in fatty livers well before associated histological changes are evident.

Androgen receptor (AR) is a transcriptional activator that, in prostate cells, stimulates gene expression required for various cellular functions, including metabolisms and proliferation. AR signaling is also essential for the development of hormone-dependent prostate cancer (PCa) and its activity can be blocked by androgen-deprivation therapies (ADTs). Although PCa patients initially respond well to ADTs, the cancer inevitably relapses and progresses to lethal castration-resistant prostate cancer (CRPC). Although AR activity is generally restored in CRPC despite the castrate level of androgens, it is unclear whether AR signaling is significantly reprogrammed. In this study, we examined the AR cistrome in a PCa cell line-derived CRPC model using integrated bioinformatical analyses. Significantly, we found that the AR cistrome is largely retained in the CRPC stage. In particular, AR-mediated lipid biosynthesis is highly conserved and reactivated during the progression to CRPC, and increased level of lipid synthesis is associated with poor prognosis. The restoration of lipid biosynthetic pathways is partially due to the increased expression of AR splice variants. Blocking lipid/cholesterol synthesis in AR variants-expressing CRPC cell line and xenograft models markedly reduces tumor growth through inhibition of mTOR pathway. Silencing the expression of a fatty acid elongase, ELOVL7, also leads to the regression of CRPC xenograft tumors. These results demonstrate the importance of reactivation of AR-regulated lipid biosynthetic pathways in driving CRPC progression, and suggest that ADTs may be therapeutically enhanced by blocking lipid biosynthetic pathways.

The standard treatment for prostate cancer (PCa) is androgen deprivation therapy (ADT) that blocks transcriptional activity of androgen receptor (AR). However, ADT invariably leads to the development of castration-resistant PCa (CRPC) with restored activity of AR. CRPC can be further treated with CYP17 inhibitors to block androgen synthesis pathways, but most patients still relapse after a year of such treatment. The mechanisms that drive this progression are not fully understood, but AR activity, at least in a subset of cancers, appears to be restored again. Importantly, AR mutations are more frequently detected in this type of cancer. By analyzing tumor biopsy mRNA from CRPC patients who had developed resistance to CYP17 inhibitor treatment, we have identified a novel nonsense mutation (Q784*) at the ligand binding domain (LBD) of AR, which produces a C-terminal truncated AR protein that lacks intact LBD. This AR-Q784* mutant is transcriptionally inactive, but it is constitutively expressed in the nucleus and can bind to DNA in the absence of androgen. Significantly, our results show that AR-Q784* can heterodimerize with, and enhance the transcriptional activity of, full-length AR. Moreover, expressing AR-Q784* in an AR positive PCa cell line enhances the chromatin binding of endogenous AR and the recruitment of p300 coactivator under the low androgen condition, leading to increased cell growth. This activity of AR-Q784* mimics the function of some AR splice variants, indicating that CYP17 inhibitor treatment in CRPC may select for LBD-truncated forms of AR to restore AR signaling.

Competing Interests: Conflicts of Interest: The authors have no conflicts of interest to declare.

Although well characterized as a transcriptional activator that drives prostate cancer (PCa) growth, androgen receptor (AR) can function as a transcriptional repressor, and high-level androgens can suppress PCa proliferation. The molecular basis for this repression activity remains to be determined. Genes required for DNA replication are highly enriched among androgen-repressed genes, and AR is recruited to the majority of these genes, where it rapidly represses their transcription. This activity is enhanced in PCa cells expressing high AR levels and is mediated by recruitment of hypophosphorylated retinoblastoma protein (Rb). Significantly, AR also indirectly increases the expression of DNA replication genes through stimulatory effects on other metabolic genes with subsequent CDK activation and Rb hyperphosphorylation. In castration-resistant PCa cells, which are dependent on high-level AR expression, this anti-proliferative repression function might be exploited through treatment with androgen in combination with agents that suppress AR-driven metabolic functions or cell cycle progression., Competing Interests: of Potential Conflicts of Interest: No potential conflicts of interest were disclosed., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Lower urinary tract symptoms (LUTS) are a range of irritative or obstructive symptoms that commonly afflict aging population. The diagnosis is mostly based on patient-reported symptoms, and current medication often fails to completely eliminate these symptoms. There is a pressing need for objective non-invasive approaches to measure symptoms and understand disease mechanisms. We developed an in-depth workflow combining urine metabolomics analysis and machine learning bioinformatics to characterize metabolic alterations and support objective diagnosis of LUTS. Machine learning feature selection and statistical tests were combined to identify candidate biomarkers, which were statistically validated with leave-one-patient-out cross-validation and absolutely quantified by selected reaction monitoring assay. Receiver operating characteristic analysis showed highly-accurate prediction power of candidate biomarkers to stratify patients into disease or non-diseased categories. The key metabolites and pathways may be possibly correlated with smooth muscle tone changes, increased collagen content, and inflammation, which have been identified as potential contributors to urinary dysfunction in humans and rodents. Periurethral tissue staining revealed a significant increase in collagen content and tissue stiffness in men with LUTS. Together, our study provides the first characterization and validation of LUTS urinary metabolites and pathways to support the future development of a urine-based diagnostic test for LUTS.

Benign prostate hyperplasia (BPH), an enlargement of the prostate common in aging in men, is associated with urinary voiding dysfunction manifest as Lower Urinary Tract Symptoms (LUTS). Although inflammation and abnormal smooth muscle contractions are known to play key roles in the development of LUTS, tissue fibrosis may also be an important and previously unrecognized contributing factor. Tissue fibrosis arises from the unregulated differentiation of fibroblasts or other precursor cell types into myofibroblasts, which is usually accomplished by activation of the TGFβ/TGFβR axis. Previously we reported that the CXC-type chemokines, CXCL5, CXCL8 and CXCL12, which are up-regulated in the aging in the prostate, can drive this differentiation process as well in the absence of TGFβ. Based on this data we sought to elucidate the molecular mechanisms employed by CXCL12, and its receptor CXCR4, during prostate myofibroblast phenoconversion. The results of these studies suggest that CXCL12/CXCR4-mediated signaling events in prostate myofibroblast phenoconversion may proceed through non-canonical pathways that do not depend on TGFβ/TGFβR axis activation or Smad signaling. Here we report that CXCL12/CXCR4 axis activation promotes signaling through the EGFR and downstream MEK/ERK and PI3K/Akt pathways during myofibroblast phenoconversion, but not through TGFβ/TGFβR and downstream Smad signaling, in prostate fibroblasts undergoing myofibroblast phenoconversion. We document that EGFR transactivation is required for CXCL12-mediated signaling and expression of genes associate with myofibroblast phenoconversion (α-SMA, COL1a1). Our study successfully identified TGFβ/TGFβR-independent molecular mechanisms that promote CXCL12/CXCR4-induced myofibroblast phenoconversion. This information may be crucial for the development of novel therapies and potential biomarkers for prostatic fibrosis.

Background: Resveratrol, a phytoalexin found in berries, peanuts, grapes, and red wine, inhibits oxidation, inflammation, and cell proliferation and collagen synthesis in multiple cell types and or animal models. It represses collagen deposition in the vasculature, heart, lung, kidney, liver, and esophagus in animal models and may have some utility as an anti-fibrotic. Recent studies have shown that increased collagen deposition and tissue stiffness in the peri-urethral area of the prostate are associated with lower urinary tract dysfunction (LUTD) and urinary obstructive symptoms. The aim of this study was to determine whether Resveratrol might be useful to inhibit or revert TGFβ- and/or CXCL12-mediated myofibroblast phenoconversion of prostate fibroblasts in vitro, and therefore whether the use of anti-fibrotic therapeutics might be efficacious for the treatment of LUTD., Methods: Primary prostate and lung tissues were explanted and fibroblast monolayers expanded in vitro. Primary and N1 immortalized prostate stromal fibroblasts, as well as primary fibroblasts cultured from a normal lung and one affected by idiopathic pulmonary fibrosis (IPF) for comparison, were grown in serum-free defined media supplemented with vehicle, TGFβ or CXCL12, pre- or post-treatment with Resveratrol, and were evaluated using immunofluorescence for alpha smooth muscle actin (αSMA) and collagen I (COL1) protein expression and assessed for cell proliferation, apoptosis, and COL1 and EGR1 transcript expression., Results: This study showed that low concentrations of Resveratrol (≤50 μM) had no effect on N1 or primary prostate fibroblast cell proliferation, apoptosis, or COL1 or EGR1 gene transcription but repressed and reversed myofibroblast phenoconversion. As expected, these same effects were observed for IPF lung fibroblasts though higher levels of Resveratrol (≥100uM) were required. Taken together, these data suggest that, like lung fibroblasts, prostate fibroblast to myofibroblast phenoconversion can be both repressed and reversed by Resveratrol treatment. Thus, anti-fibrotic therapeutics might be efficacious for the treatment of LUTD.

A key difference between normal and malignant prostate cells in vitro and in vivo is that both alleles of PTEN are largely intact in normal benign prostate glands and cultured epithelial cells, whereas one or both alleles of PTEN are mutant or deleted in the majority of prostate tumors and malignant prostate cancer cell lines. Intact PTEN suppresses phosphorylation of Akt downstream of PI3K activation in non-transformed cells whereas Akt phosphorylation is unimpeded in malignant cells that are often PTEN-deficient. We have previously shown that activation of the CXCL12/CXCR4 axis transactivates the EGFR to promote pro-proliferative signaling preferentially through the Raf/MEK/Erk pathway in benign prostate epithelial cells. These cells demonstrate little basal pAkt and these levels do not increase with CXCL12 stimulation because PTEN is intact and fully functional. Thus, inactivation of PTEN may be the critical factor that modulates downstream signaling and the specific CXCL12-stimulated proliferative responses of non-transformed and transformed prostate epithelial cells. Based on these data, we hypothesize that the CXCL12/CXCR4-mediated activation of downstream pro-proliferative signaling through the Raf/MEK/Erk or PI3K/Akt pathways is modulated by PTEN status.

Solitary fibrous tumors (SFTs) of the prostate are a rare type of spindle cell neoplasm that can demonstrate either a benign or malignant phenotype. SFTs represent a clinical challenge along with other spindle cell lesions of the prostate in terms of both diagnosis and treatment. The present study shows, for the first time, that SFTs of the prostate and other organs can comprise a mixed population of fibroblast, myofibroblast, and smooth muscle cell types. The highly proliferative component demonstrated a fibroblastic phenotype that readily underwent myofibroblast differentiation on exposure to profibrotic stimuli. Consistent with other recent studies, the prostatic SFTs demonstrated NAB2-STAT6 gene fusions that were also present in the fibroblast, myofibroblast, and smooth muscle cell types of the SFT. The results of these studies suggest that benign and malignant prostatic tumors of mesenchymal origin may be distinguished at the molecular and cellular levels, and that delineation of such defining characteristics may help elucidate the etiology and prognosis of such tumors., (Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Benign prostatic hyperplasia (BPH) is a major health concern for aging men. BPH is associated with urinary voiding dysfunction and lower urinary tract symptoms (LUTS), which negatively affects quality of life. Surgical resection and medical approaches have proven effective for improving urinary flow and relieving LUTS but are not effective for all men and can produce adverse effects that require termination of the therapeutic regimen. Thus, there is a need to explore other therapeutic targets to treat BPH/LUTS. Complicating the treatment of BPH/LUTS is the lack of biomarkers to effectively identify pathobiologies contributing to BPH/LUTS or to gauge successful response to therapy. This review will briefly discuss current knowledge and will highlight new studies that illuminate the pathobiologies contributing to BPH/LUTS, potential new therapeutic strategies for successfully treating BPH/LUTS, and new approaches for better defining these pathobiologies and response to therapeutics through the development of biomarkers and phenotyping strategies.

Lower urinary tract symptoms (LUTS)--constituting a spectrum disorder that encompasses weak stream, nocturia, and sensations of incomplete emptying and intermittent or hesitant urination--are indicative of lower urinary tract dysfunction (LUTD). LUTD is a progressive disease that can lead to bladder dysfunction if left untreated or treated ineffectively. Sequelae include urinary retention, recurrent UTI, bladder calculi, and, eventually, renal impairment. LUTD involving the prostate is associated with both ageing and inflammation. Tissue inflammation resulting from ageing, infection, or other inflammatory disease processes (for example, type 2 diabetes mellitus) is epidemiologically associated with the subsequent development of tissue fibrosis in multiple organ systems, including the prostate. Recent studies show that tissue fibrosis in the lower urinary tract is associated with LUTD, and suggest that fibrosis might be a previously unrecognized pathobiology that contributes to LUTD. Thus, antifibrotic therapeutic agents should be considered as a new approach to efficaciously treating men with LUTD, especially those who don't experience durable responses to 5α-reductase inhibitors or α-adrenergic receptor antagonists.

Background: Progressive aging- and inflammation-associated fibrosis effectively remodels the extracellular matrix (ECM) to increase prostate tissue stiffness and reduce urethral flexibility, resulting in urinary flow obstruction and lower urinary tract symptoms (LUTS). In the current study, we sought to test whether senescence-accelerated mouse prone (SAMP)6 mice, which were reported to develop prostatic fibrosis, would also develop LUTS, and whether these symptoms would be exacerbated by diet-induced obesity and concurrent Type 2 Diabetes Mellitus (T2DM)., Methods: To accomplish this, SAMP6 and AKR/J background strain mice were fed regular mouse chow, low fat diet chow, or high fat diet chow for 8 months, then subjected to glucose tolerance tests, assessed for plasma insulin levels, evaluated for urinary voiding function, and assessed for lower urinary tract fibrosis., Results: The results of these studies show that SAMP6 mice and AKR/J background strain mice develop diet-induced obesity and T2DM concurrent with urinary voiding dysfunction. Moreover, urinary voiding dysfunction was more severe in SAMP6 than AKR/J mice and was associated with pronounced prostatic and urethral tissue fibrosis., Conclusions: Taken together, these studies suggest that obesity, T2DM, lower urinary tract fibrosis, and urinary voiding dysfunction are inextricably and biologically linked., (Copyright © 2013 Wiley Periodicals, Inc.)

Background: The recent recommendation of the U.S. Preventive Services Task Force against PSA-based screening for prostate cancer was based, in part, on the lack of demonstrated diagnostic utility of serum PSA values in the low, but detectable range to successfully predict prostate cancer. Though controversial, this recommendation reinforced the critical need to develop, validate, and determine the utility of other serum and/or urine transcript and protein markers as diagnostic markers for PCa. The studies described here were intended to determine whether inflammatory cytokines might augment serum PSA as a diagnostic marker for prostate cancer., Methods: Multiplex ELISA assays were performed to quantify CCL1, CCL2, CCL5, CCL8, CCL11, CCL17, CXCL1, CXCL5, CXCL8, CXCL10, CXCL12, and IL-6 protein levels in the serum of 272 men demonstrating serum PSA values of <10 ng/ml and undergoing a 12 core diagnostic needle biopsy for detection of prostate cancer. Logistic regression was used to identify the associations between specific chemokines and prostate cancer status adjusted for prostate volume, and baseline PSA., Results: Serum levels for CCL1 (I-309) were significantly elevated among all men with enlarged prostates (P < 0.04). Serum levels for CCL11 (Eotaxin-1) were significantly elevated among men with prostate cancer regardless of prostate size (P < 0.01). The remaining 10 cytokines examined in this study did not exhibit significant correlations with either prostate volume or cancer status., Conclusions: Serum CCL11 values may provide a useful diagnostic tool to help distinguish between prostatic enlargement and prostate cancer among men demonstrating low, but detectable, serum PSA values., (Copyright © 2012 Wiley Periodicals, Inc.)

Circulating tumor cells (CTCs) detached from both primary and metastatic lesions represent a potential alternative to invasive biopsies as a source of tumor tissue for the detection, characterization and monitoring of cancers. Here we report a simple yet effective strategy for capturing CTCs without using capture antibodies. Our method uniquely utilized the differential adhesion preference of cancer cells to nanorough surfaces when compared to normal blood cells and thus did not depend on their physical size or surface protein expression, a significant advantage as compared to other existing CTC capture techniques.

Purpose: Current therapies for male lower urinary tract symptoms secondary to prostate enlargement prevent hormonal effects on prostate growth and inhibit smooth muscle contraction to ease bladder neck and urethral pressure. However, lower urinary tract symptoms can be refractory to these therapies, suggesting that additional biological processes not addressed by them may also contribute to lower urinary tract symptoms. Aging associated fibrotic changes in tissue architecture contribute to dysfunction in multiple organ systems. Thus, we tested whether such changes potentially have a role in impaired urethral function and perhaps in male lower urinary tract symptoms., Materials and Methods: Periurethral tissues were obtained from a whole prostate ex vivo and from 28 consecutive men treated with radical prostatectomy. Lower urinary tract symptoms were assessed using the American Urological Association symptom index. Prostate tissues were subjected to mechanical testing to assess rigidity and stiffness. Fixed sections of these tissues were evaluated for collagen and elastin content, and glandularity to assess fibrosis. Statistical analysis included the Student t test and calculation of Pearson correlation coefficients to compare groups., Results: Periurethral prostate tissues demonstrated nonlinear viscoelastic mechanical behavior. Tissue from men with lower urinary tract symptoms was significantly stiffer (p = 0.0016) with significantly higher collagen content (p = 0.0038) and lower glandularity than that from men without lower urinary tract symptoms (American Urological Association symptom index 8 or greater vs 7 or less)., Conclusions: Findings show that extracellular matrix deposition and fibrosis characterize the periurethral prostate tissue of some men with lower urinary tract symptoms. They point to fibrosis as a factor contributing to lower urinary tract symptom etiology., (Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

The molecular mechanisms responsible for the transition of some prostate cancers from androgen ligand-dependent to androgen ligand-independent are incompletely established. Molecules that are ligands for G protein coupled receptors (GPCRs) have been implicated in ligand-independent androgen receptor (AR) activation. The purpose of this study was to examine whether CXCL12, the ligand for the GPCR, CXCR4, might mediate prostate cancer cell proliferation through AR-dependent mechanisms involving functional transactivation of the AR in the absence of androgen. The results of these studies showed that activation of the CXCL12/CXCR4 axis promoted: The nuclear accumulation of both wild-type and mutant AR in several prostate epithelial cell lines; AR-dependent proliferative responses; nuclear accumulation of the AR co-regulator SRC-1 protein; SRC-1:AR protein:protein association; co-localization of AR and SRC-1 on the promoters of AR-regulated genes; AR- and SRC-1 dependent transcription of AR-regulated genes; AR-dependent secretion of the AR-regulated PSA protein; P13K-dependent phosphorylation of AR; MAPK-dependent phosphorylation of SRC-1, and both MAPK- and P13K-dependent secretion of the PSA protein, in the absence of androgen. Taken together, these studies identify CXCL12 as a novel, non-steroidal growth factor that promotes the growth of prostate epithelial cells through AR-dependent mechanisms in the absence of steroid hormones. These findings support the development of novel therapeutics targeting the CXCL12/CXCR4 axis as an ancillary to those targeting the androgen/AR axis to effectively treat castration resistant/recurrent prostate tumors., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Recent studies from our group suggest that extracellular matrix (ECM) deposition and fibrosis characterize the peri-urethral prostate tissues of some men suffering from Lower Urinary Tract Symptoms (LUTS) and that fibrosis may be a contributing factor to the etiology of LUTS. Fibrosis can generally be regarded as an errant wound-healing process in response to chronic inflammation, and several studies have shown that the aging prostate tissue microenvironment is rich with inflammatory cells and proteins. However, it is unclear whether these same inflammatory proteins, particularly CXC-type chemokines, can mediate myofibroblast phenoconversion and the ECM deposition necessary for the development of prostatic tissue fibrosis. To examine this, immortalized and primary prostate stromal fibroblasts treated with TGF-β1, CXCL5, CXCL8, or CXCL12 were evaluated morphologically by microscopy, by immunofluorescence and qRT-PCR for αSMA, collagen 1, vimentin, calponin, and tenascin protein and transcript expression, and by gel contraction assays for functional myofibroblast phenoconversion. The results of these studies showed that that immortalized and primary prostate stromal fibroblasts are induced to express collagen 1 and 3 and αSMA gene transcripts and proteins and to undergo complete and functional myofibroblast phenoconversion in response to CXC-type chemokines, even in the absence of exogenous TGF-β1. Moreover, CXCL12-mediated myofibroblast phenoconversion can be completely abrogated by inhibition of the CXCL12 receptor, CXCR4. These findings suggest that CXC-type chemokines, which comprise inflammatory proteins known to be highly expressed in the aging prostate, can efficiently and completely mediate myofibroblast phenoconversion and may thereby promote fibrotic changes in prostate tissue architecture associated with the development and progression of male lower urinary tract dysfunction.

The tumor microenvironment (TME) is a very complex niche that consists of multiple cell types, supportive matrix and soluble factors. Cells in the TME consist of both host cells that are present at tumor site at the onset of tumor growth and cells that are recruited in either response to tumor- or host-derived factors. PCa (PCa) thrives on crosstalk between tumor cells and the TME. Crosstalk results in an orchestrated evolution of both the tumor and microenvironment as the tumor progresses. The TME reacts to PCa-produced soluble factors as well as direct interaction with PCa cells. In return, the TME produces soluble factors, structural support and direct contact interactions that influence the establishment and progression of PCa. In this review, we focus on the host side of the equation to provide a foundation for understanding how different aspects of the TME contribute to PCa progression. We discuss immune effector cells, specialized niches, such as the vascular and bone marrow, and several key protein factors that mediate host effects on PCa. This discussion highlights the concept that the TME offers a potentially very fertile target for PCa therapy.

A wealth of published studies indicate that a variety of chemokines are actively secreted by the prostatic microenvironment consequent to disruptions in normal tissue homeostasis due to the aging process or inflammatory responses. The accumulation of senescent stromal fibroblasts, and, possibly, epithelial cells, may serve as potential driving forces behind chemokine secretion in the aging and enlarged human prostate. Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) and histological inflammation may also potentially serve as rich sources of chemokine secretion in the prostate. Once bound to their cognate receptors, chemokines can stimulate powerful pro-proliferation signal transduction pathways and thus function as potent growth factors in the development and progression of Benign Prostatic Hyperplasia (BPH) and lower urinary tract symptoms (LUTS). These functions have been amply demonstrated experimentally and particularly point to robust Mitogen Activated Protein Kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signaling, as well as global transcriptional responses, which mediate chemokine-stimulated cellular proliferative responses. A small body of literature also suggests that chemokine-mediated angiogenesis may comprise a contributing factor to BPH/LUTS development and progression. Thus, the observed low-level secretion of multiple chemokines within the aging prostatic microenvironment may promote a concomitant low-level, but cumulative, over-proliferation of both stromal fibroblastic and epithelial cell types associated with increased prostatic volume. Though the accumulated evidence is far from complete and suffers from some rather extensive gaps in knowledge, it argues favorably for the conclusion that chemokines can, and likely do, promote prostatic enlargement and the associated lower urinary tract symptoms, and justifies further investigations examining chemokines as potential therapeutic targets to delay or ablate BPH/LUTS initiation and progression., (Copyright © 2011 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Background: Histological evidence of pervasive inflammatory infiltrate has been noted in both benign prostatic hyperplasia/hypertrophy (BPH) and prostate cancer (PCa). Cytokines known to attract particular leukocyte subsets are secreted from prostatic stroma consequent to aging and also from malignant prostate epithelium. Therefore, we hypothesized that leukocytes associated with either acute or chronic inflammation attracted to the prostate consequent to aging or tumorigenesis may promote the abnormal cellular proliferation associated with BPH and PCa., Methods: An in vitro system designed to mimic the human prostatic microenvironment incorporating prostatic stroma (primary and immortalized prostate stromal fibroblasts), epithelium (N15C6, BPH-1, LNCaP, and PC3 cells), and inflammatory infiltrate (HL-60 cells, HH, and Molt-3 T-lymphocytes) was developed. Modified Boyden chamber assays were used to test the ability of prostate stromal and epithelial cells to attract leukocytes and to test the effect of leukocytes on prostate cellular proliferation. Antibody arrays were used to identify leukocyte-secreted cytokines mediating prostate cellular proliferation., Results: Leukocytic cells migrated towards both prostate stromal and epithelial cells. CD4+ T-lymphocytes promoted the proliferation of both transformed and non-transformed prostate epithelial cell lines tested, whereas CD8+ T-lymphocytes as well as dHL-60M macrophagic and dHL-60N neutrophilic cells selectively promoted the proliferation of PCa cells., Conclusions: The results of these studies show that inflammatory cells can be attracted to the prostate tissue microenvironment and can selectively promote the proliferation of non-transformed or transformed prostate epithelial cells, and are consistent with differential role(s) for inflammatory infiltrate in the etiologies of benign and malignant proliferative disease in the prostate., (Prostate 70: 377-389, 2010. (c) 2009 Wiley-Liss, Inc.)

Introduction: The ectodomain shedding of epidermal growth factor receptor (EGFR) ligands, such as amphiregulin (AREG), by ADAMs (A Disintegrin And Metalloproteases) can be stimulated by G protein-coupled receptor (GPCR) agonists. Interactions between the CXCR4 GPCR and the CXCL12 chemokine have been shown to mediate gene transcription and cellular proliferation in non-transformed and transformed prostate epithelial cells, as well as motility/invasiveness in transformed cells., Objectives: In this report, we investigated the ability of CXCL12 to stimulate amphiregulin ectodomain shedding in non-transformed and transformed prostate epithelial cells that respond proliferatively to sub-nanomolar levels of CXCL12 and amphiregulin., Materials and Methods: Non-transformed N15C6 and transformed PC3 prostate epithelial cells were assessed for amphiregulin shedding, ADAM activation, Src phosphorylation and EGFR activation using ELISA, immunoblot, and immunoprecipitation techniques, and for proliferation using cell counting after stimulation with CXCL12 or vehicle., Results: The results of these studies identify CXCL12 as a novel inducer of amphiregulin ectodomain shedding and show that both basal and CXCL12-mediated amphiregulin shedding are ADAM10- and Src kinase-dependent in non-transformed N15C6 cells. In contrast, amphiregulin shedding is not amplified subsequent to stimulation with exogenous CXCL12, and is not reduced subsequent to metalloprotease- or Src kinase-inhibition, in highly aggressive PC3 prostate cancer cells. These data also show that CXCL12-mediated cellular proliferation requires EGFR transactivation in a Src- and ADAM-dependent manner in non-transformed prostate epithelial cells. However, these same mechanisms are dysfunctional in highly transformed prostate cancer cells, which secrete amphiregulin in an autocrine manner that cannot be repressed through metalloprotease- or Src kinase inhibition., Conclusion: These findings show that non-transformed and transformed prostate epithelial cells may employ different mechanisms to activate EGFR ligands and thereby utilize the EGFR axis to promote cellular proliferation.

Dysregulation of Bcl2 family member proteins has been associated with poor chemotherapeutic response in bladder cancer, suggesting that agents targeting these crucial proteins may provide an interventional strategy to slow or halt bladder cancer progression and metastasis. In this study, we investigated whether the cottonseed polyphenol, -(-)gossypol, a BH3 mimetic, can reduce the expression of pro-survival, or increase the expression of pro-apoptotic, Bcl2 family proteins and thereby effectively sensitize otherwise resistant bladder cancer cells to the standard chemotherapeutic drugs gemcitabine, paclitaxel and carboplatin. These studies show that gossypol induced apoptosis in both chemosensitive UM-UC2 and chemoresistant resistant UM-UC9 bladder cancer cells in vitro in a dose and time dependent manner via a caspase mediated death signaling pathway. Moreover, in combined treatments, gossypol synergized with gemcitabine and carboplatin to induce apoptosis in chemoresistant bladder cancer cells. This effect was associated with the down-regulation the Bcl-xl and Mcl-1 pro-survival Bcl2 family proteins and up-regulation of the Bim and Puma BH3-only Bcl2 family proteins. Overall, these studies show that gossypol sensitizes bladder cancer cells to standard chemotherapeutic drugs and may provide a promising new strategy for bladder cancer treatment.

An unbiased cDNA expression phage library derived from bone-marrow endothelial cells was used to identify novel surface adhesion molecules that might participate in metastasis. Herein we report that reticulocalbin 1 (RCN1) is a cell surface-associated protein on both endothelial (EC) and prostate cancer (PCa) cell lines. RCN1 is an H/KDEL protein with six EF-hand, calcium-binding motifs, found in the endoplasmic reticulum. Our data indicate that RCN1 also is expressed on the cell surface of several endothelial cell lines, including human dermal microvascular endothelial cells (HDMVECs), bone marrow endothelial cells (BMEC), and transformed human bone marrow endothelial cells (TrHBMEC). While RCN1 protein levels were highest in lysates from HDMVEC, this difference was not statistically significant compared BMEC and TrHBMEC. Given preferential adhesion of PCa to bone-marrow EC, these data suggest that RCN1 is unlikely to account for the preferential metastasis of PCa to bone. In addition, there was not a statistically significant difference in total RCN1 protein expression among the PCa cell lines. RCN1 also was expressed on the surface of several PCa cell lines, including those of the LNCaP human PCa progression model and the highly metastatic PC-3 cell line. Interestingly, RCN1 expression on the cell surface was upregulated by tumor necrosis factor alpha treatment of bone-marrow endothelial cells. Taken together, we show cell surface localization of RCN1 that has not been described previously for either PCa or BMEC and that the surface expression on BMEC is regulated by pro-inflammatory TNF-alpha.