Adult Prostate Research Articles

Slug-expressing mouse prostate epithelial cells have increased stem cell potential.

Deciphering the properties of adult stem cells is crucial for understanding of their role in healthy tissue and in cancer progression as well. Both stem cells and cancer stem cells have shown association with epithelial-to-mesenchymal transition (EMT) in various tissue types. Aiming to investigate the epithelial and mesenchymal phenotypic traits in adult mouse prostate, we sorted subpopulations of basal prostate stem cells (mPSCs) and assessed the expression levels of EMT regulators and markers with custom-designed gene expression array. The population of mPSCs defined by a Lin-/Sca-1+CD49fhi/Trop-2+ (LSC Trop-2+) surface phenotype was enriched in mesenchymal markers, especially EMT master regulator Slug, encoded by the Snai2 gene. To further dissect the role of Slug in mPSCs, we used transgenic Snai2tm1.1Wbg reporter mouse strain. Using this model, we confirmed the presence of mesenchymal traits and increase of organoid forming capacity in Slug+ population of mPSCs. The Slug+-derived organoids comprised all prostate epithelial cell types - basal, luminal, and neuroendocrine. Collectively, these data uncover the important role of Slug expression in the physiology of mouse prostate stem cells.

SUN-139 Luminescence-Based Drug Screen for Novel Androgen Receptor Antagonists for Prostate Cancer Therapy

The development and maintenance of the adult prostate is dependent on the action of androgens, which mediate its effect by binding to the androgen receptor (AR). Dysregulation in the AR signaling axis disrupts transcriptional homeostasis, shifting the balance towards uncontrolled proliferation and driving the progression towards prostate cancer (PCa). The prominent role of AR signaling in prostate carcinogenesis led to the development of androgen deprivation therapy (ADT) as the primary treatment strategy for managing PCa. Despite the success of ADT in early PCa cases, most patients develop resistance to ADT and the cancer ultimately recurs towards a lethal state termed castration-resistant prostate cancer (CRPC). Remarkably, AR signaling is still active in CRPC, suggesting that the progression towards CRPC is still reliant on AR activity. Current treatments are ineffective against CRPC, highlighting the need for alternative therapeutics that can combat the resistant nature of CRPC. To address the need for innovative approaches against CRPC, we used a robust luminescence-based bioassay that can identify novel AR antagonists. We screened plant extracts derived from local fauna by measuring their effect on AR-driven activity using a luciferase-based reporter assay in HeLa cells stably overexpressing hAR (HeLa-hAR). To identify candidate hits, HeLa-hAR cells were treated with DHT to induce AR-dependent luciferase activity, DHT with the AR antagonist bicalutamide as a positive control, and DHT plus the plant extracts. We identified one extract, A32, which showed significant inhibition of AR-dependent luciferase activity without having deleterious effects on cell viability. Secondary validation tests also showed that A32 exhibits a dose-dependent inhibition of AR-driven reporter activity. When testing the effect of A32 on gene expression in LNCaP cells, we observed a down-regulation in the expression of canonical AR target genes such as PSA to degrees similar to bicalutamide. These results suggest that A32 may be an AR antagonist or may target the AR signaling axis. Collectively, this study establishes the use of a luminescence-based reporter assay for the identification of novel AR antagonists from a plant extract library.

Rhabdomyosarcoma of the Adult Prostate: Two Cases With Molecular and Cytogenetic Analyses

Abstract Primary rhabdomyosarcoma (RMS) of the adult prostate is a very rare tumor with only 45 cases published to date. The clinical course of RMS of the prostate is very aggressive, and prognosis is very poor. Here we describe two cases of primary RMS of the prostate of adult patients and discuss the differential diagnosis of RMS with other mesenchymal tumors of prostate. The first patient was a 50-year-old man who clinically presented with urinary retention and hematuria, low serum prostate-specific antigen, moderate prostatomegaly, and multiple metastases as shown by computed tomography. Histological examination revealed a diffuse “small round blue cell” proliferation with an intermingled population of larger cells with rhabdomyoblastic differentiation that showed immunoreactivity for desmin and MYF4 (myogenin). Molecular and cytogenetic studies did not reveal recurrent chromosomal translocations associated with RMSs and other sarcomas. The patient underwent doxorubicin (adriamycin) treatment without disease progression during the whole follow-up period of 7 months. The second patient was a 39-year-old man who presented with urinary retention, an increased serum prostate-specific antigen level, extensive prostatomegaly, enlarged abdominal and inguinal lymph nodes, and skeletal metastases. Histological examination revealed a tumoral proliferation with a small round blue aspect and diffuse immunoreactivity for desmin and MYF4 (myogenin). Fluorescence in situ hybridization revealed a FOXO1 translocation, consistent with a diagnosis of alveolar-type RMS. The patient followed a multimodal treatment and died of the disease after its progression.

MP06-01 IDENTIFICATION OF COGNATE PROXIMAL CELL TYPES OF THE MOUSE AND HUMAN PROSTATE AND THEIR ENRICHMENT IN HUMAN BENIGN PROSTATIC HYPERPLASIA

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research & Pathophysiology (MP06)1 Apr 2020MP06-01 IDENTIFICATION OF COGNATE PROXIMAL CELL TYPES OF THE MOUSE AND HUMAN PROSTATE AND THEIR ENRICHMENT IN HUMAN BENIGN PROSTATIC HYPERPLASIA Diya Binoy Joseph, Gervaise Henry, Alicia Malewska, Kyle Wegner, Jeffrey Reese, Ryan Mauck, Jeffrey Gahan, Ryan Hutchinson, Claus Roehrborn, Chad Vezina, and Douglas Strand* Diya Binoy JosephDiya Binoy Joseph More articles by this author , Gervaise HenryGervaise Henry More articles by this author , Alicia MalewskaAlicia Malewska More articles by this author , Kyle WegnerKyle Wegner More articles by this author , Jeffrey ReeseJeffrey Reese More articles by this author , Ryan MauckRyan Mauck More articles by this author , Jeffrey GahanJeffrey Gahan More articles by this author , Ryan HutchinsonRyan Hutchinson More articles by this author , Claus RoehrbornClaus Roehrborn More articles by this author , Chad VezinaChad Vezina More articles by this author , and Douglas Strand*Douglas Strand* More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000820.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Benign prostatic hyperplasia (BPH) is highly prevalent in aging men and poses a significant healthcare burden associated with the treatment of Lower Urinary Tract Symptoms (LUTS). BPH/LUTS remain difficult to treat because of phenotypic heterogeneity, resulting in the need for surgical intervention. The cell types that originate BPH growth are not known. Anatomical studies have shown that the transition zone located near the urethra is the site of BPH growth. We previously identified two novel cell types, club and hillock, that are enriched in the prostatic urethra and proximal ducts. Here, we identify a new proximal fibroblast in the human as well as cognate hillock epithelia and proximal fibroblasts in the mouse. We assess the contribution of each proximal cell type in human BPH, establishing a new paradigm for urethral epithelia and proximal fibroblasts in discrete BPH phenotypes. METHODS: We used an unbiased approach by single cell RNA-sequencing (scRNA-seq) to identify cognate cell types of the mouse and human prostate and develop new flow cytometry and IHC antibody panels to purify and locate each cell type. The frequency of proximal fibroblasts and urethral epithelia in BPH vs. normal human prostate was assessed using scRNA-seq, flow cytometry, and immunofluorescence. RESULTS: Hillock and club cell identity is established early and these cells extend into the proximal ducts of the adult prostate transition zone. Proximal fibroblasts surround the urethra and proximal ducts. BPH patients have increased club and hillock cells within glandular nodules compared to normal prostate tissue and proximal fibroblasts are increased in areas of peri-urethral fibrosis. Mouse scRNA-seq and IHC data confirmed the existence of cognate proximal fibroblasts and hillock epithelia, but club cells were not found. CONCLUSIONS: Our results show that club and hillock epithelia of the urethra and proximal ducts are established before prostate budding and are enriched in BPH glandular nodules, suggesting a potential cellular origin for new prostate growth. We also identify the proximal fibroblast of the human as the cellular source of collagen deposition in prostatic fibrosis, setting up a new phenotype to target in patients with LUTS. We created a cellular atlas of the mouse urethra and proximal prostate, which will allow for the generation of specialized mouse models to trace urethral and proximal fibroblast lineages. The cell types of the proximal ducts could become novel targets for the treatment of BPH/LUTS. Source of Funding: R01 DK115477 © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e51-e51 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Diya Binoy Joseph More articles by this author Gervaise Henry More articles by this author Alicia Malewska More articles by this author Kyle Wegner More articles by this author Jeffrey Reese More articles by this author Ryan Mauck More articles by this author Jeffrey Gahan More articles by this author Ryan Hutchinson More articles by this author Claus Roehrborn More articles by this author Chad Vezina More articles by this author Douglas Strand* More articles by this author Expand All Advertisement PDF downloadLoading ...

Epidemiology and survival outcome of adult kidney, bladder, and prostate rhabdomyosarcoma: A SEER database analysis.

Rhabdomyosarcoma (RMS) is rare in adulthood, accounting for 2%–5% of adult soft tissue tumors, and less than 20% occur in genitourinary organs. Given its rarity, survival data on adult kidney, bladder, and prostate RMSs is limited. In this population-based analysis, we performed an analysis of all adult RMS cases reported in Surveillance, Epidemiology, and End Results (SEER) database to understand prognostic factors among kidney, bladder, and prostate RMS. A query of the SEER database was performed from 1973 to 2016 for patients >18 of age with RMS. The final cohort consisted of 14 kidney, 35 bladder, and 21 prostate RMS cases in the adult population. Demographic, treatment, and survival data were obtained. Analysis was performed using Fisher’s exact test, survival analysis, and model. The median (range) age of diagnosis for adult bladder RMS was 65 years old (19–84) compared to 52.5 (28–68) and 42 (19–87) for kidney and prostate (p = 0.007). About 78.6% of patients underwent surgical intervention. Five-year overall survival (OS) for adult kidney, bladder, and prostate RMS are 17.1% (2.9–41.6%), 22.2% (9.4–38.4%), and 33.0 (12.8–55.0%), respectively. OS was not statistically associated with primary site (p = 0.209). On multivariable analysis, compared to adult bladder RMS, kidney RMS had a higher incidence of mortality (HR: 2.16, 95% CI 1.03–4.53, p = 0.041). Incidence of mortality from prostate RMS was not significantly different from bladder RMS (HR: 0.70, 95% CI 0.30–1.65, p = 0.411). Extent of disease (HR: 5.17, 95% CI 2.09–12.79, p < 0.001) and older age (HR 1.03, 95% CI 1.01–1.04, p = 0.002) were adverse prognostic factors for OS. Overall survival at 5 years for adult kidney, bladder, and prostate RMS is poor. Localized disease and younger age are prognostic factors for improved outcomes in adult RMS. Hence, early diagnosis and intervention appear paramount to improved survival for this rare malignancy in adulthood.

The risk of developing cardiovascular disease is increased for patients with prostate cancer who are pharmaceutically treated for depression.

ObjectiveTo examine the associations between pharmaceutically treated anxiety and depression and incident cardiovascular disease (CVD) among 1‐year prostate cancer survivors.Patients and methodsA registry‐based cohort study design was used to describe the risk of incident CVD in adult 1‐year prostate cancer survivors without a history of CVD. Patients with prostate cancer diagnosed between 1999 and 2011 were selected from the Netherlands Cancer Registry. Drug dispenses were retrieved from the PHARMO Database Network and were used as proxy for CVD, anxiety, and depression. Data were analysed using Cox regression analysis to examine the risk associations between pharmaceutically treated anxiety and depression entered as a time‐varying predictor with incident CVD in 1‐year prostate cancer survivors, while controlling for age, traditional CVD risk factors, and clinical characteristics.ResultsOf the 5262 prostate cancer survivors, 327 (6%) developed CVD during the 13‐year follow‐up period. Prostate cancer survivors who were pharmaceutically treated for depression had an increased risk of incident CVD after full adjustment compared to prostate cancer survivors who were not pharmaceutically treated for depression (hazard ratio [HR] 1.51, 95% confidence interval [CI] 1.06–2.15). The increased risk of incident CVD amongst those pharmaceutically treated for depression compared to those who were not pharmaceutically treated for depression, was only valid among: prostate cancer survivors who were aged ≤65 years (HR 2.91; 95% CI 1.52–5.55); those who were not treated with radiotherapy (HR 1.63; 95% CI 1.01–2.65); those who were treated with hormones (HR 1.76; 95% CI 1.09–2.85); those who were not operated upon (HR 1.55; 95% CI 1.07–2.25); and those with tumour stage III (HR 2.21; 95% CI 1.03–4.74) and stage IV (HR 2.47; 95% CI 1.03–5.89).ConclusionPatients with prostate cancer who were pharmaceutically treated for depression had a 51% increased risk of incident CVD after adjustment for anxiety, age, traditional CVD risk factors, and clinical characteristics. The results emphasise the need to pay attention to (pharmaceutically treated) depressed patients with prostate cancer prior to deciding on prostate cancer treatment and for a timely detection and treatment of CVD.

Mechanisms of decision-making in preoperative assessment for older adult prostate cancer patients-A qualitative study.

Little research exists which investigates the contextual factors and hidden influences that inform surgeons and surgical teams decision-making in preoperative assessment when deciding whether to or not to operate on older adult prostate cancer patients living with aging-associated functional declines and illnesses. The aim of this study is to identify and examine the underlying mechanisms that uniquely shape preoperative surgical decision-making strategies concerning older adult prostate cancer patients. Qualitative methodologies were used that paired ethnographic field observations with semistructured interviews for data collection. An inductive thematic analysis approach was used to identify, analyze, and describe patterns in the data. Factors underlining surgical decision-making originated from the context of two categories: (1) clinical and surgery-specific factors; and (2) non-patient factors. Thematic subcategories included personal experiences, methods of assessment during medical encounters, anticipation of outcomes, perceptions of preoperative assessment instruments for frailty and multimorbidity, routines and workflow patterns, microcultures, and indirect observation and second-hand knowledge. Surgeon's personal experiences has a significant impact on the decision-making processes during preoperative assessments. However, non-patient factors such as institutional microcultures passively and actively influence decision-making process during preoperative assessment.

Heparan sulfate inhibits transforming growth factor β signaling and functions in cis and in trans to regulate prostate stem/progenitor cell activities.

Prostate stem/progenitor cells (PrSCs) are responsible for adult prostate tissue homeostasis and regeneration. However, the related regulatory mechanisms are not completely understood. In this study, we examined the role of heparan sulfate (HS) in PrSC self-renewal and prostate regeneration. Using an in vitro prostate sphere formation assay, we found that deletion of the glycosyltransferase exostosin 1 (Ext1) abolished HS expression in PrSCs and disrupted their ability to self-renew. In associated studies, we observed that HS loss inhibited p63 and CK5 expression, reduced the number of p63+- or CK5+-expressing stem/progenitor cells, elevated CK8+ expression and the number of differentiated CK8+ luminal cells and arrested the spheroid cells in the G1/G0 phase of cell cycle. Mechanistically, HS expressed by PrSCs (in cis) or by neighboring cells (in trans) could maintain sphere formation. Furthermore, HS deficiency upregulated transforming growth factor β (TGFβ) signaling and inhibiting TGFβ signaling partially restored the sphere-formation activity of the HS-deficient PrSCs. In an in vivo prostate regeneration assay, simultaneous loss of HS in both epithelial cell and stromal cell compartments attenuated prostate tissue regeneration, whereas the retention of HS expression in either of the two cellular compartments was sufficient to sustain prostate tissue regeneration. We conclude that HS preserves self-renewal of adult PrSCs by inhibiting TGFβ signaling and functions both in cis and in trans to maintain prostate homeostasis and to support prostate regeneration.

Differences between male and female prostates in terms of physiology, sensitivity to chemicals and pathogenesis-A review in a rodent model.

The prostate is a gland that is not exclusively present in males, being also found in females of several mammalian species, including humans. There is evidence that the prostate in both sexes is affected by the same pathologies such as prostatitis, benign alterations and even cancer. In view of the difficulties of manipulating the prostate gland, the Mongolian gerbil (Meriones unguiculatus), a rodent species with high incidence of functional prostates in females, is widely used in studies of the female prostate. However, despite knowing much about the similarities between the female and male prostate, little emphasis has been placed on the differences between them. This review investigates the intersex differences in prostate development, physiology and pathogenesis. The female prostate develops earlier than in males and studies indicate that it is more sensitive to oestrogens than the male prostate, as well as being more sensitive to exposure to xenoestrogens, such as Bisphenol A and methylparaben, with a higher susceptibility to benign lesions in the adult and senile prostate than in males. In addition, the female prostate is impacted by pregnancy and the oestrous cycle, and is also dependent on progesterone. The peculiarities of the female prostate raise concerns about the risk of it undergoing neglected changes as a result of environmental chemicals, since safe dosages are established exclusively for the male prostate.

Abstract 5261: Choline kinase downregulation decreases prostate cancer associated fibroblast viability

Abstract Introduction We previously observed an increase of prostate CAFs (PCAFs) in prostate cancers that metastasize, consistent with the known role of fibroblasts in inducing growth, confer castration-resistance and increasing metastatic potential. While aberrant choline metabolism has been identified in prostate cancer cells, choline metabolism in fibroblasts and PCAFs is unexplored and may identify a treatment strategy through downregulation of enzymes such as choline kinase alpha (Chk-α), the enzyme that converts choline to phosphocholine (PC). Chk-α is typically increased in cancer cells. Here we characterized the metabolic profile of normal prostate fibroblasts and PCAFs as well as determined the effect of Chk-α downregulation with small interfering RNA (siRNA). Methods Experiments were performed using the human prostate myofibroblasts (WPMY-1, derived from stromal cells from the peripheral zone of the histologically normal adult prostate) and PCAFs (obtained from an adenocarcinoma of the prostate gland). Cell extracts were obtained using a dual-phase extraction method. High-resolution 1H MR spectra were recorded on a Bruker Biospin Avance-III 750 MHz NMR (Bruker Biospin) spectrometer operating at a 1H frequency of 750.21 MHz using a 5-mm broad band inverse (BBI) probe head equipped with z-gradient accessories. 1H MR spectra were manually phased and automated baseline corrected using TOPSPIN 3.2 software. Integrals of the metabolites of interest were determined and normalized to the TSP reference and the number of cells. Metabolites were estimated from at least three experimental samples. Statistical significance was evaluated using the Student’s t-test. To target Chk-α, cells were transiently transfected with small interfering RNA (siRNA) against Chk-α at a concentration of 100 nM for 48h using standard protocol. Total RNA was isolated, complementary cDNA synthesized and quantitative real-time PCR performed using IQ SYBR Green supermix and gene specific primers. Viability studies were performed using CCK8 assay as previously reported. Results and Discussion Metabolic profiles were significantly different between WPMY-1 and PCAFs, with significant differences in choline, phosphocholine, glutamate and alanine, among others. siRNA directed against Chk-α reduced mRNA levels in both WPMY-1 and PCAFs. We observed a significant decrease of cell viability in PCAFs but not in normal prostate fibroblasts. These data support investigating Chk-α as a target to eliminate CAFs in tumors. Increased glutamate observed in PCAFs also support targeting enzymes and transporters in glutamine/glutamate metabolism as potential therapeutic strategies against PCAFs. Future studies with CAFs from different cancers should further validate the metabolic differences between normal fibroblasts and CAFs identified in this study. Supported by NIH R35CA209960. JPT is supported by Martin-Escudero Foundation. Citation Format: Jesus Pacheco-Torres, Tariq Shah, Flonne Wildes, Balaji Krishnamachary, Zaver M. Bhujwalla. Choline kinase downregulation decreases prostate cancer associated fibroblast viability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5261.

SAT-328 Androgens Modulate the Expression of Prostatic Acid Phosphatase

INTRODUCTION: Prostatic acid phosphatase (PAP) is an acid phosphatase synthesized in prostate epithelial cells that served as the biochemical diagnostic mainstay for prostate cancer for several decades. There are two forms of PAP, secretory(sPAP) and transmembrane (TM-PAP), that are derived from alternative transcripts of the gene. PAP is highly expressed by epithelial cells of the adult prostate, however, the androgenic regulation of prostatic acid phosphatase is not well understood. HYPOTHESIS AND OBJECTIVES: We hypothesized that androgens do not increase PAP expression. Our objectives were to determine the immunohistochemical expression of PAP in human fetal prostate tissue, human bone metastases, and intact and castrated xenograft tumors of human VCaP prostate cancer cells. In addition, the effects of dihydrotestosterone (DHT) onTM- and sPAP expression in vitro were quantified. METHODS: Immunohistochemical staining for PAP, androgen receptor (AR) and Prostate Specific Antigen (PSA) protein expression was carried out on human fetal prostate (9.5, 11.5, 13, 16.5 and 18 weeks of gestational age) and human castration-resistant prostate cancer bone metastases archival specimens obtained with IRB approval. Tissue from human benign prostate and breast cancer metastases were used as controls. For xenograft studies, VCaP cells were inoculated subcutaneously into SCID mice and surgical or sham castration was performed after tumor development. Mouse tumors and weights were measured biweekly and tumor tissue isolated after mouse sacrifice. Isolated tumors were stained for PAP, AR and PSA. RESULTS: PAP expression was an early marker of prostate development and observed in the fetal prostate as early as 11.5 weeks of gestational age. Strong PAP expression was observed in all human castration-resistant prostate cancer bone metastases examined (4/4 specimens). The AR was absent in two of four specimens. PSA was expressed in human castration-resistant bone metastatic prostate specimens; however, the expression was not as strong as PAP. In vivo, castrated VCaP tumors underwent tumor stasis and were significantly smaller compared to intact animals but increased PAP expression was noted after castration. In contrast, PSA and AR expression was reduced in castrated VCaP tumors compared to tumors from intact mice. DHT treatment of VCaP cells increased AR expression while decreasing both sPAP and TM-PAP at doses ranging from 10-7-10-14M for 24 h. CONCLUSIONS: Our findings demonstrate that PAP is expressed early in normal human fetal prostate development and may be a marker of the early stages of differentiation. DHT decreased the expression of both isoforms of PAP in vitro and castration increased PAP expression in vivo. TM-PAP may serve as a potential target for castration-resistant prostate cancer as its expression increases with castration.

A temporal and spatial map of axons in developing mouse prostate.

Prostate autonomic and sensory axons control glandular growth, fluid secretion, and smooth muscle contraction and are remodeled during cancer and inflammation. Morphogenetic signaling pathways reawakened during disease progression may drive this axon remodeling. These pathways are linked to proliferative activities in prostate cancer and benign prostate hyperplasia. However, little is known about which developmental signaling pathways guide axon investment into prostate. The first step in defining these pathways is pinpointing when axon subtypes first appear in prostate. We accomplished this by immunohistochemically mapping three axon subtypes (noradrenergic, cholinergic, and peptidergic) during fetal, neonatal, and adult stages of mouse prostate development. We devised a method for peri-prostatic axon density quantification and tested whether innervation is uniform across the proximo-distal axis of dorsal and ventral adult mouse prostate. Many axons directly interact with or innervate neuroendocrine cells in other organs, so we examined whether sensory or autonomic axons innervate neuroendocrine cells in prostate. We first detected noradrenergic, cholinergic, and peptidergic axons in prostate at embryonic day (E) 14.5. Noradrenergic and cholinergic axon densities are uniform across the proximal-distal axis of adult mouse prostate while peptidergic axons are denser in the periurethral and proximal regions. Peptidergic and cholinergic axons are closely associated with prostate neuroendocrine cells whereas noradrenergic axons are not. These results provide a foundation for understanding mouse prostatic axon development and organization and, provide strategies for quantifying axons during progression of prostate disease.

A pivotal role of androgen signaling in Notch-responsive cells in prostate development, maturation, and regeneration

Androgen signaling is essential for prostate development, morphogenesis, and regeneration. Emerging evidence also indicates a regulatory role of Notch signaling in prostate development, differentiation, and growth. However, the collaborative regulatory mechanisms of androgen and Notch signaling during prostate development, growth, and regeneration are largely unknown. Hairy and Enhancer of Split 1 (Hes1) is a transcriptional regulator of Notch signaling pathways, and its expression is responsive to Notch signaling. Hes1-expressing cells have been shown to possess the regenerative capability to repopulate a variety of adult tissues. In this study, we developed new mouse models to directly assess the role of the androgen receptor in prostatic Hes1-expressing cells. Selective deletion of AR expression in embryonic Hes1-expressing cells impeded early prostate development both in vivo and in tissue xenograft experiments. Prepubescent deletion of AR expression in Hes1-expressing cells resulted in prostate glands containing abnormalities in cell morphology and gland architecture. A population of castration-resistant Hes1-expressing cells was revealed in the adult prostate, with the ability to repopulate prostate epithelium following androgen supplementation. Deletion of AR in Hes1-expressing cells diminishes their regenerative ability. These lines of evidence demonstrate a critical role for the AR in Notch-responsive cells during the course of prostate development, morphogenesis, and regeneration, and implicate a mechanism underlying interaction between the androgen and Notch signaling pathways in the mouse prostate.

Functional Heterogeneity of Mouse Prostate Stromal Cells Revealed by Single-Cell RNA-Seq

SummaryWe perform a single-cell RNA sequencing analysis to investigate the phenotypic and functional heterogeneity of the adult mouse prostate stromal cells. Our analysis identifies three major cell populations representing the smooth muscle cells and two types of fibroblast cells enriched by Sca-1 and CD90. The Sca-1+CD90+ fibroblast cells are in direct contact with the epithelial cells and express growth factors and genes associated with cell motility, developmental process, and androgen biosynthesis. This suggests that they may regulate epithelial cell survival and growth. The Sca-1+CD90-/low myofibroblast-like cells highly express genes associated with the extracellular matrix and cytokine-mediated signaling pathways, indicating a role in tissue repair and immune responses. The Sca-1+CD90-/low cells significantly suppress the capacity of the basal cells for bipotent differentiation in the prostate organoid assay. Collectively, we identify the surface markers enabling physical separation of stromal subpopulations and generate the gene expression profiles implying their cellular functions.

Sox2 Expression Marks Castration-Resistant Progenitor Cells in the Adult Murine Prostate.

Identification of defined epithelial cell populations with progenitor properties is critical for understanding prostatic development and disease. Here, we demonstrate that Sox2 expression is enriched in the epithelial cells of the proximal prostate adjacent to the urethra. We use lineage tracing of Sox2-positive cells during prostatic development, homeostasis, and regeneration to show that the Sox2 lineage is capable of self-renewal and contributes to prostatic regeneration. Persisting luminal cells express Sox2 after castration, highlighting a potential role for Sox2 in cell survival and castration-resistance. In addition to revealing a novel progenitor population in the prostate, these data implicate Sox2 as a regulatory factor of adult prostate epithelial stem cells. Stem Cells 2019;37:690-700.

Epithelial DNA methyltransferase-1 regulates cell survival, growth and maturation in developing prostatic buds

DNA methyltransferase 1 (DNMT1) is required for embryogenesis but roles in late forming organ systems including the prostate, which emerges from the urethral epithelium, have not been fully examined. We used a targeted genetic approach involving a Shhcre recombinase to demonstrate requirement of epithelial DNA methyltransferase-1 (Dnmt1) in mouse prostate morphogenesis. Dnmt1 mutant urethral cells exhibit DNA hypomethylation, DNA damage, p53 accumulation and undergo cell cycle arrest and apoptosis. Urethral epithelial cells are disorganized in Dnmt1 mutants, leading to impaired prostate growth and maturation and failed glandular development. We evaluated oriented cell division as a mechanism of bud elongation and widening by demonstrating that mitotic spindle axes typically form parallel or perpendicular to prostatic bud elongation axes. We then deployed a ShhcreERT allele to delete Dnmt1 from a subset of urethral epithelial cells, creating mosaic mutants with which to interrogate the requirement for cell division in specific prostatic bud epithelial populations. DNMT1- cell distribution within prostatic buds is not random as would be expected in a process where DNMT1 was not required. Instead, replication competent DNMT1 + cells primarily accumulate in prostatic bud margins and tips while replication impeded DNMT1- cells accumulate in prostatic bud cores. Together, these results highlight the role of DNMT1 in regulating epithelial bud formation by maintaining cell cycle progression and survival of rapidly dividing urethral epithelial cells, which can be extended to the study of other developing epithelial organs. In addition, our results show that prostatic buds consist of two epithelial cell populations with distinct molecular and functional characteristics that could potentially contribute to specialized lineages in the adult prostate.

Edar is a downstream target of beta-catenin and drives collagen accumulation in the mouse prostate.

ABSTRACTBeta-catenin (CTNNB1) directs ectodermal appendage spacing by activating ectodysplasin A receptor (EDAR) transcription, but whether CTNNB1 acts by a similar mechanism in the prostate, an endoderm-derived tissue, is unclear. Here we examined the expression, function, and CTNNB1 dependence of the EDAR pathway during prostate development. In situ hybridization studies reveal EDAR pathway components including Wnt10b in the developing prostate and localize these factors to prostatic bud epithelium where CTNNB1 target genes are co-expressed. We used a genetic approach to ectopically activate CTNNB1 in developing mouse prostate and observed focal increases in Edar and Wnt10b mRNAs. We also used a genetic approach to test the prostatic consequences of activating or inhibiting Edar expression. Edar overexpression does not visibly alter prostatic bud formation or branching morphogenesis, and Edar expression is not necessary for either of these events. However, Edar overexpression is associated with an abnormally thick and collagen-rich stroma in adult mouse prostates. These results support CTNNB1 as a transcriptional activator of Edar and Wnt10b in the developing prostate and demonstrate Edar is not only important for ectodermal appendage patterning but also influences collagen organization in adult prostates.This article has an associated First Person interview with the first author of the paper.

Luminal-contact-inhibition of epithelial basal stem cell multipotency in prostate organogenesis and homeostasis.

ABSTRACTProstate epithelial basal cells are highly plastic in their luminal differentiation capability. Basal stem cells actively produce luminal cells during organogenesis, but become restricted in the adult prostate unless receiving oncogenic or inflammatory stimuli. Given that the number of luminal cells increases relative to basal cells through development and that equilibrium is reached in the adulthood, we hypothesize that a negative-feedback mechanism exists to inhibit basal-to-luminal differentiation. We provide evidence supporting this hypothesis by comparing murine prostatic growth in a tissue reconstitution assay with cell recombinants of different basal-to-luminal ratios. Additionally, in organoid culture, hybrid organoids derived from adjacent basal and luminal cells showed reduced basal stem cell activities, suggesting contact inhibition. Importantly, removal of adult luminal cells in vivo via either an inducible Cre/loxP-Dre/rox dual-lineage-tracing system or orthotopic trypsin injection led to robust reactivation of basal stem cell activities, which acts independent of androgen. These data illustrate the prostate organ as a distinctive paradigm where cell contact from differentiated daughter cells restricts adult stem cell multipotency to maintain the steady-state epithelial architecture.

Cells of Origin for Prostate Cancer.

Comprehensive knowledge of the normal prostate epithelial lineage hierarchy is a prerequisite to investigate the identity of the cells of origin for prostate cancer. The basal and luminal cells constitute most of the prostate epithelium and have been the major focuses of the study on the cells of origin for prostate cancer. Much progress has been made during the past few decades, mainly using mouse models, to understand the inter-lineage relationship and intra-lineage heterogeneity in adults as well as the lineage plasticity during conditions of stress. These studies have concluded that the adult mouse prostate basal and luminal cells are largely independently sustained under physiological conditions, but both types of cells possess the capacity for bipotent differentiation under stress or artificial experimental conditions. However, the existence or the identity of the putative progenitors within each lineage warrants further investigation. Whether the human prostate lineage hierarchy is completely the same as that of the mouse remains uncertain. Experiments from independent groups have demonstrated that both types of cells in mice and humans can serve as targets for transformation. But controversies remain whether the disease from distinct cells of origin display different clinical behaviors. Further investigation of the intra-lineage heterogeneity will provide new insights into this issue. Understanding the identity of the cells of origin for prostate cancer will help identify novel prognostic markers for early detection of aggressive prostate cancers, provide insights into the therapeutic vulnerability of these tumors, and inspire novel therapeutic strategies.

A Cellular Anatomy of the Normal Adult Human Prostate and Prostatic Urethra

SUMMARYA comprehensive cellular anatomy of normal human prostate is essential for solving the cellular origins of benign prostatic hyperplasia and prostate cancer. The tools used to analyze the contribution of individual cell types are not robust. We provide a cellular atlas of the young adult human prostate and prostatic urethra using an iterative process of single-cell RNA sequencing (scRNA-seq) and flow cytometry on ~98,000 cells taken from different anatomical regions. Immunohistochemistry with newly derived cell type-specific markers revealed the distribution of each epithelial and stromal cell type on whole mounts, revising our understanding of zonal anatomy. Based on discovered cell surface markers, flow cytometry antibody panels were designed to improve the purification of each cell type, with each gate confirmed by scRNA-seq. The molecular classification, anatomical distribution, and purification tools for each cell type in the human prostate create a powerful resource for experimental design in human prostate disease.