Adult Human Prostate Research Articles

Abstract 5323: Inflammation drives lineage plasticity and a club cell-like phenotype in prostate epithelial cells

Abstract Background: Club cells are a type of bronchiolar epithelial cell that serve a protective and antimicrobial function in the lung. An epithelial cell population in adult human prostates was recently identified as sharing a similar transcriptomic profile and morphology to lung club cells. Club-like epithelial cells were identified in the prostatic urethra, collecting ducts, and rarely in the peripheral zone of normal organ donor prostates. However, we recently reported that cells expressing club cell markers (CP, LTF, MMP7, PIGR, SCGB1A1) are present in the peripheral zone of radical prostatectomy specimens. Here, expression of club cell markers was restricted to luminal epithelial cells in an inflammation-associated lesion termed proliferative inflammatory atrophy (PIA). We also demonstrated that these club-like cells within PIA express the same markers as intermediate cells, a proposed prostate cancer progenitor cell. We now aim to identify exogenous factors that drive the club cell phenotype in vitro. Our ultimate goal is to understand how club cell gene expression affects the oncogenic and metastatic potential of prostate epithelial cells. Methods: We characterized the expression of club cell genes in prostate cell lines (LNCaP, PREC, and PREC-AR-Myc) following exposure to inflammatory stimuli (TNFα, hydrogen peroxide, uropathogenic Escherichia coli) in vitro. We performed quantitative reverse transcription PCR (RT-qPCR) to measure expression of club cell markers (LTF, MMP7, PIGR) and intermediate cell markers (AR, NKX3.1, SMOX). Additionally, we used chromogenic RNA in situ hybridization (RISH) to visualize LTF, MMP7, and PIGR expression following exposure to inflammatory stimuli. Results: We report that TNFα exposure in vitro increased expression of club cell genes (LTF, MMP7, PIGR). Surprisingly, TNFα exposure induced LTF expression in LNCaP cells, despite prior findings that LTF is silenced by CpG island hypermethylation in prostate cancer and in LNCaP cells. This finding was confirmed by both RT-qPCR and RISH. Additionally, we found that TNFα exposure altered intermediate cell markers (AR, NKX3.1, SMOX) expression consistent with the previously reported gene expression pattern of luminal epithelial cells in PIA. Conclusions: Overall, our results suggest that TNFα-mediated signaling drives the expression of club cell markers associated with lineage plasticity. Our next step will be to analyze inflammation-driven alterations to methylation and chromatin accessibility around loci associated with club cell genes, proton-oncogenes, and tumor suppressor genes. Citation Format: Megan Hess, Hanbing Song, Jessica Hicks, Luke Mummert, Angelo De Marzo, Franklin W. Huang, Karen Sfanos. Inflammation drives lineage plasticity and a club cell-like phenotype in prostate epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5323.

Club-like cells in proliferative inflammatory atrophy of the prostate.

Club cells are a type of bronchiolar epithelial cell that serve a protective role in the lung and regenerate damaged lung epithelium. Single-cell RNA sequencing (scRNA-seq) of young adult human prostate and urethra identified cell populations in the prostatic urethra and collecting ducts similar in morphology and transcriptomic profile to lung club cells. We further identified club cell-like epithelial cells by scRNA-seq of prostate peripheral zone tissues. Here, we aimed to identify and spatially localize club cells in situ in the prostate, including in the peripheral zone. We performed chromogenic RNA in situ hybridization for five club cell markers (CP, LTF, MMP7, PIGR, SCGB1A1) in a series of (1) nondiseased organ donor prostate and (2) radical prostatectomy specimens from individuals with prostate cancer. We report that expression of club cell genes in the peripheral zone is associated with inflammation and limited to luminal epithelial cells classified as intermediate cells in proliferative inflammatory atrophy (PIA). Club-like cells were enriched in radical prostatectomy specimens compared to nondiseased prostates and associated with high-grade prostate cancer. We previously reported that luminal epithelial cells in PIA can rarely harbor oncogenic TMPRSS2:ERG (ERG+) gene fusions, and we now demonstrate that club cells are present in association with ERG+ PIA that is transitioning to early adenocarcinoma. Finally, prostate epithelial organoids derived from prostatectomy specimens demonstrate that club-like epithelial cells can be established in organoids and are sensitive to anti-androgen-directed treatment in vitro in terms of decreased androgen signaling gene expression signatures compared to basal or hillock cells. Overall, our study identifies a population of club-like cells in PIA and proposes that these cells play an analogous role to that of club cells in bronchiolar epithelium. Our results further suggest that inflammation drives lineage plasticity in the human prostate and that club cells in PIA may be prone to oncogenic transformation. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

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.

Correlation between 5-α reductase type 2 protein expression and methylation of 5-α reductase type 2 promotor gene of benign prostatic hyperplasia.

The enzyme 5-α reductase type 2 (5-AR 2) plays a key role in the development and maintenance of the prostate gland. We evaluated the level 5-AR 2 protein expression and the relationship between methylation of the 5-AR 2 gene-promoter and 5-AR 2 protein expression of benign prostatic hyperplasia (BPH). A total of 37 prostate samples were evaluated. These included 22 samples from men undergoing transurethral prostate resections and 15 non-cancerous transition-zone human prostate tissue samples taken following radical prostatectomy. We quantified 5-AR 2 protein expression and gene-promoter methylation status using common assay procedures. Clinical variables included age, body mass index (BMI), prostate-specific antigen (PSA) levels, lipid profiles, and prostate volumes. Univariate and multivariate statistical analyses were performed followed by stepwise logistic regression modeling. We were able to extract DNA from 36 of the 37 tissue samples and 10 of these (28%) did not express the 5-AR 2 protein. In total, 26 patients (72%) had methylated 5-AR 2 promoter-regions. There was a strong correlation between methylation of the 5-AR 2 promoter-regions and low-absent 5-AR 2 protein expression (p = 0.0003). Increasing age significantly predicted methylation status and protein expression level (p = 0.013). The level of 5-AR 2 protein expression varies among prostate tissue samples. Methylation of the 5-AR 2 gene-promoter may account for low or absent expression of 5-AR 2 in adult human prostate tissues. Increased age correlates with increased 5-AR 2 gene-promoter methylation and decreased protein expression in men with BPH.

Androgenic to oestrogenic switch in the human adult prostate gland is regulated by epigenetic silencing of steroid 5α-reductase 2.

Benign prostatic hyperplasia is the most common proliferative abnormality of the prostate. All men experience some prostatic growth as they age, but the rate of growth varies among individuals. Steroid 5α-reductase 2 (SRD5A2) is a critical enzyme for prostatic development and growth. Previous work indicates that one-third of adult prostatic samples do not express SRD5A2, secondary to epigenetic modifications. Here we show that the level of oestradiol is dramatically elevated, concomitant with significant upregulation of oestrogen response genes, in prostatic samples with methylation at the SRD5A2 promoter. The phosphorylation of oestrogen receptor-α in prostatic stroma is upregulated when SRD5A2 expression is absent. We show that tumour necrosis factor (TNF)-α suppresses SRD5A2 mRNA and protein expression, and simultaneously promotes expression of aromatase, the enzyme responsible for conversion of testosterone to oestradiol. Concomitant suppression of SRD5A2 and treatment with TNF-α synergistically upregulate the aromatase levels. The data suggest that, in the absence of prostatic SRD5A2, there is an androgenic to oestrogenic switch. These findings have broad implications for choosing appropriate classes of medications for the management of benign and malignant prostatic diseases. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Multipotent Basal Stem Cells, Maintained in Localized Proximal Niches, Support Directed Long-Ranging Epithelial Flows in Human Prostates

SummarySporadic mitochondrial DNA mutations serve as clonal marks providing access to the identity and lineage potential of stem cells within human tissues. By combining quantitative clonal mapping with 3D reconstruction of adult human prostates, we show that multipotent basal stem cells, confined to discrete niches in juxta-urethral ducts, generate bipotent basal progenitors in directed epithelial migration streams. Basal progenitors are then dispersed throughout the entire glandular network, dividing and differentiating to replenish the loss of apoptotic luminal cells. Rare lineage-restricted luminal stem cells, and their progeny, are confined to proximal ducts and provide only minor contribution to epithelial homeostasis. In situ cell capture from clonal maps identified delta homolog 1 (DLK1) enrichment of basal stem cells, which was validated in functional spheroid assays. This study establishes significant insights into niche organization and function of prostate stem and progenitor cells, with implications for disease.

Isolation and functional interrogation of adult human prostate epithelial stem cells at single cell resolution

Using primary cultures of normal human prostate epithelial cells, we developed a novel prostasphere-based, label-retention assay that permits identification and isolation of stem cells at a single cell level. Their bona fide stem cell nature was corroborated using in vitro and in vivo regenerative assays and documentation of symmetric/asymmetric division. Robust WNT10B and KRT13 levels without E-cadherin or KRT14 staining distinguished individual stem cells from daughter progenitors in spheroids. Following FACS to isolate label-retaining stem cells from label-free progenitors, RNA-seq identified unique gene signatures for the separate populations which may serve as useful biomarkers. Knockdown of KRT13 or PRAC1 reduced sphere formation and symmetric self-renewal highlighting their role in stem cell maintenance. Pathways analysis identified ribosome biogenesis and membrane estrogen-receptor signaling enriched in stem cells with NF-ĸB signaling enriched in progenitors; activities that were biologically confirmed. Further, bioassays identified heightened autophagy flux and reduced metabolism in stem cells relative to progenitors. These approaches similarly identified stem-like cells from prostate cancer specimens and prostate, breast and colon cancer cell lines suggesting wide applicability. Together, the present studies isolate and identify unique characteristics of normal human prostate stem cells and uncover processes that maintain stem cell homeostasis in the prostate gland.

Epigenetic silencing and variable expression of SRD5A2 in specific compartments of human prostate.

38 Background: The steroid-5α reductase type 2 gene (SRD5A2) and protein play a significant role in the development and growth of prostate tissue. SRD5A2 can be expressed in both the epithelial and stromal compartments of the prostate gland. Previously, we have shown that somatic suppression of SRD5A2 during adulthood is dependent on epigenetic changes associated with methylation of the promoter region of the SRD5A2 gene. Here we determined whether variability of SRD5A2 expression occurs in specific compartments of prostate tissues. Methods: Frozen and formalin-fixed sections of human adult prostate tissue was used. Laser capture microdissection (LCM) was performed to obtain a pure population of epithelial and stromal cells. Approximately 500–600 excised cells were captured, and the methylation pattern of SRD5A2 was determined by DNA methylation pull-down assay kit. The expression of SRD5A2 protein was identified with immunohistochemistry and quantitated by ELISA. Results: In the whole prostate tissue, samples with high SRD5A2 immunoreactivity had much lower levels of SRD5A2 promoter methylation compared to those with low SRD5A2 immunoreactivity. DNA methylation assay for the epithelial compartment closely resembled the findings of the whole prostate tissue. In contrast, the samples from the stromal compartment showed uniform methylation irrespective of the methylation pattern in the epithelial compartment or the whole tissue. The expression of SRD5A2 protein quantitated by ELISA further showed that the variable expression of SRD5A2 is better determined by epithelial compartment expression. Conclusions: Our data suggests that variability of SRD5A2 expression is dependent on the methylation pattern and expression of SRD5A2 in the epithelial compartment and not the stroma. Therefore, if epithelial expression of SRD5A2 is more representative of the whole prostatic tissue expression, then it is logical that SRD5A2 inhibitor’s efficacy is dependent on involution of the epithelial cells. Our findings will have broad implications for the use of 5ARIs in the treatment of BPH and for the chemoprevention of prostate cancer. Grant support: NIH/R01 DK091353

Regulation of steroid-5-alpha-reductase 2 (SRD5A2) in human prostate by epigenetic modifications.

204 Background: 5-alpha reductase type 2 (SRD5A2), an enzyme that is critical for prostatic development and growth. We have found that many aging prostate tissues do not express the enzyme. We previously showed that expression of SRD5A2 is not static and epigenetic modulations by DNA methyltransferase and pro-inflammatory cytokines play important roles in silencing of SRD5A2. Here we wished to define the methylation pattern and identify specific CpG dinucleotides that are methylated in the SRD5A2promoter region. Methods: Ninety six prostate samples from patients were obtained by transurethral resection of prostate (TURP). Methylation of SRD5A2 promoter was assessed using Methylated CpG Island Recovery Assay (MIRA). Bisulfide conversion of Genomic DNA was performed using Epimark bisulfide conversion kit. Primers for bisulfide sequencing were designed using MethPrimer software and bisulfide sequencing was performed using 3730XL DNA Sequencers. Binding of histone demethylase to promoter DNA was analyzed by ChiP assay. Results: We randomly selected 3 TURP samples with methylated SRD5A2 promoters and 3 TURP samples with un-methylated SRD5A2 promoters, and then bisulfide conversion and sequencing were performed to verify the methylation pattern of CpG dinucleotides on SRD5A2 promoter. We found that bisulfide sequencing analysis of SRD5A2 promoter was consistent with MIRA analysis. To further analyze the mediators of SRD5A2 promoter region, we show that TNF-alpha up-regulates expression of Snail protein, which is a central regulator of both DNA methylation and histone methylation, and increases the binding of H3K9me to SRD5A2promoter region, but not H3K27me or H4K20me. Conclusions: Methylation of SRD5A2 promoter, which is regulated by DNMT1, proinflammatory cytokines and the histone methylase, H3K9me, accounts for suppression of SRD5A2 in many adult human prostate tissues. Bisulfide conversion and sequencing confirm and focus the specified CpG dinucleotides that are methylated enabling us to evaluate mechanisms and patterns of SRD5A2 promoter methylation in order to study the functional significance of SRD5A2 methylation. Grant support: NIH/R01 DK091353.

Effects of Extreme Dilutions of Apis mellifica Preparations on Gene Expression Profiles of Human Cells.

Gene expression analysis has been employed in the past to test the effects of high dilutions on cell systems. However, most of the previous studies were restricted to the investigation of few dilutions, making it difficult to explore underlying mechanisms of action. Using whole-genome transcriptomic analysis, we investigated the effects of a wide range of Apis mellifica dilutions on gene expression profiles of human cells. RWPE-1 cells, a nonneoplastic adult human epithelial prostate cell line, were exposed to Apis mellifica preparations (3C, 5C, 7C, 9C, 12C, 15C, and 30C) or to the reference solvent solutions for 24 hours; nonexposed cells were also checked for gene expression variations. Our results showed that even the most diluted solutions retained the ability to trigger significant variations in gene expression. Gene pathway analysis revealed consistent variations in gene expression induced by Apis mellifica when compared to nonexposed reference cells but not to reference solvent solutions. Since the effects of Apis Mellifica at extreme dilutions did not show dose–effect relationships, the biological or functional interpretation of these results remains uncertain.

Organoid model shows effect of BPA on prostate development.

Studies using rodent and adult human prostate stem-progenitor cell models indicate that exposure to bisphenol A (BPA) during development increases susceptibility of the prostate gland to premalignancy later in life. A novel human organoid model has now been generated to study the effects of BPA on embryonic human prostate development.

Abstract 1052: Variable expression of 5-alpha reductase 2 in the aging adult prostate is regulated by DNA methylation

Abstract BACKGROUND: 5-alpha reductase type 2 (SRD5A2), an enzyme that is critical for prostatic development and growth, is utilized as an inhibitory target by finasteride for patients with bladder outlet obstrution secondary to BPH. However, we have found that many aging benign prostate tissues do not express the enzyme. Since the SRD5A2 promoter contains a CpG island, we hypothesized that somatic methylation of the promoter would be regulated by DNA methyltransferases leading to suppression of SRD5A2. METHODS: Benign prostatic tissues from wild-type mice at 3, 6 and 12 months of age were used. In addition, 96 prostate samples from human male patients who were treated by TURP for bladder outlet obstruction secondary to BPH were used. Methylation of SRD5A2 promoter was assessed using Methylated CpG Island Recovery Assay (MIRA). DNMT1 siRNA and 5-AZA-C were used to determine the methylation status of SRD5A2 in benign prostatic cell line, BPH-1. To determine the effect of CpG methylation, SRD5A2 promoter-luciferase constructs were methylated in vitro using M.SssI methylase. Statistical analyses were performed with JMP Pro version 11 (SAS Institute Inc., Cary, NC). RESULTS: We analyzed 96 human BPH samples from transurethral resection of prostate (TURP) surgeries and found that absence of SRD5A2 was closely associated with hypermethylation of the SRD5A2 promoter region. We found that methylation of SRD5A2 was regulated by DNA methytransferase 1 (DNMT1) and the cytokines, TNF-alpha, NF-κB and IL-6, regulated DNMT1protein expression and thereby indirectly affected SRD5A2 promoter methylation and gene expression. Suppression of cytokines inhibited activity of DNMT1, while over-expression of p65 or treatment with TNF-alpha or IL-6 up-regulated DNMT1. In addition, we found that methylation of the SRD5A2 promoter and concomitant decrease in protein expression were closely associated with patient's increasing age (P<0.05). Finally, in murine prostate tissues, cytokine levels, DNMT1 and global methylation were all increased in older mice. Induction of inflammation with lipopolysaccharide (LPS) stimulated the TNFR1/NF-κB/IL-6/DNMT1 pathway, leading to hypermethylation of the SRD5A2 promoter and silencing of SRD5A2, while treatment with both LPS and TNF-alpha inhibitor reversed this pathway and reactivated SRD5A2. CONCLUSIONS: Expression of SRD5A2 is not static and ubiquitous in benign adult prostate tissues. We show that SRD5A2 expression is lacking in many benign human adult prostate tissues. Methylation of SRD5A2 promoter region, which is regulated by DNMT1, accounts for absence of SRD5A2 expression in many adult human prostate tissues. Methylation and expression of SRD5A2 may be used as a gene signature to tailor therapies for more effective treatment of prostatic diseases. Citation Format: Ge Rongbin, Zongwei Wang, Seth Bechis, Alexander Otsetov, Shengyu Hua, Shulin Wu, Chin-Lee Wu, Shahin Tabatabaei, Aria Olumi. Variable expression of 5-alpha reductase 2 in the aging adult prostate is regulated by DNA methylation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1052. doi:10.1158/1538-7445.AM2015-1052

Directed Differentiation of Human Embryonic Stem Cells into Prostate Organoids In Vitro and its Perturbation by Low-Dose Bisphenol A Exposure.

Studies using rodent and adult human prostate stem-progenitor cell models suggest that developmental exposure to the endocrine disruptor Bisphenol-A (BPA) can predispose to prostate carcinogenesis with aging. Unknown at present is whether the embryonic human prostate is equally susceptible to BPA during its natural developmental window. To address this unmet need, we herein report the construction of a pioneer in vitro human prostate developmental model to study the effects of BPA. The directed differentiation of human embryonic stem cells (hESC) into prostatic organoids in a spatial system was accomplished with precise temporal control of growth factors and steroids. Activin-induced definitive endoderm was driven to prostate specification by combined exposure to WNT10B and FGF10. Matrigel culture for 20–30 days in medium containing R-Spondin-1, Noggin, EGF, retinoic acid and testosterone was sufficient for mature prostate organoid development. Immunofluorescence and gene expression analysis confirmed that organoids exhibited cytodifferentiation and functional properties of the human prostate. Exposure to 1 nM or 10 nM BPA throughout differentiation culture disturbed early morphogenesis in a dose-dependent manner with 1 nM BPA increasing and 10 nM BPA reducing the number of branched structures formed. While differentiation of branched structures to mature organoids seemed largely unaffected by BPA exposure, the stem-like cell population increased, appearing as focal stem cell nests that have not properly entered lineage commitment rather than the rare isolated stem cells found in normally differentiated structures. These findings provide the first direct evidence that low-dose BPA exposure targets hESC and perturbs morphogenesis as the embryonic cells differentiate towards human prostate organoids, suggesting that the developing human prostate may be susceptible to disruption by in utero BPA exposures.

RUNX1 is essential for mesenchymal stem cell proliferation and myofibroblast differentiation.

Myofibroblasts are a key cell type in wound repair, cardiovascular disease, and fibrosis and in the tumor-promoting microenvironment. The high accumulation of myofibroblasts in reactive stroma is predictive of the rate of cancer progression in many different tumors, yet the cell types of origin and the mechanisms that regulate proliferation and differentiation are unknown. We report here, for the first time to our knowledge, the characterization of normal human prostate-derived mesenchymal stem cells (MSCs) and the TGF-β1-regulated pathways that modulate MSC proliferation and myofibroblast differentiation. Human prostate MSCs combined with prostate cancer cells expressing TGF-β1 resulted in commitment to myofibroblasts. TGF-β1-regulated runt-related transcription factor 1 (RUNX1) was required for cell cycle progression and proliferation of progenitors. RUNX1 also inhibited, yet did not block, differentiation. Knockdown of RUNX1 in prostate or bone marrow-derived MSCs resulted in cell cycle arrest, attenuated proliferation, and constitutive differentiation to myofibroblasts. These data show that RUNX1 is a key transcription factor for MSC proliferation and cell fate commitment in myofibroblast differentiation. This work also shows that the normal human prostate gland contains tissue-derived MSCs that exhibit multilineage differentiation similar to bone marrow-derived MSCs. Targeting RUNX1 pathways may represent a therapeutic approach to affect myofibroblast proliferation and biology in multiple disease states.

Abstract 2313: Dynamic expression of 5-alpha reductase 2 in aging prostate is regulated by DNA methyltransferase 1

Abstract BACKGROUND: 5-alpha reductase type 2 (SRD5A2), an enzyme that is critical for prostatic development and growth, is utilized as an inhibitory target by Finasteride for patients with BPH. However, we have found that many aging benign prostate tissues do not express the enzyme. Since the SRD5A2 promoter contains a CpG island, we hypothesized that somatic methylation of the promoter may account for absence of SRD5A2 expression. METHODS: Benign prostatic tissues from wild-type mice at 3, 6 and 12 months of age were used. In addition, prostate samples from human male patients who were treated by TURP for bladder outlet obstruction secondary to BPH were used. Methylation of SRD5A2 promoter was assessed using Methylated CpG Island Recovery Assay (MIRA). DNMT1 siRNA and 5-AZA-C were used to determine the methylation status of SRD5A2 in benign prostatic cells (BPH-1). SRD5A2 promoter-luciferase constructs were methylated in vitro using M.SssI methylase. RESULTS: Benign prostatic tissues from wild-type mice of different ages (3 months, 6 months and 12 months, n=6 per group) were harvested. We found that expression of SRD5A2 and DNMT1, 3a, 3b were not significantly changed in mice at the age of 3 and 6 months. However, 33% of mice at age of 12 months (2/6) had low levels of SRD5A2 with concomitant high DNMT1 protein levels. Consistent with reduced expression of SRD5A2 and increased DNMT1, the methylation of SRD5A2 promoter was increased as assayed by MIRA. Moreover, silencing DNMT1 with siRNA or exposure of BPH-1 cells to 5-AZA-C led to re-expression of SRD5A2. We used SRD5A2 promoter-luciferase constructs that were methylated in vitro using M.SssI methylase. When the methylated SRD5A2 promoter constructs were ectopically expressed in BPH-1 cells, we found that methylation of the SRD5A2 promoter caused reduction of luciferase expression by 80%, suggesting that methylation of the SRD5A2 promoter regulates the expression of SRD5A2. Since age-associated increased IL-6 has been shown to regulate DNMT1, we next evaluated expression of IL-6 in aging prostate tissue. We found that expression of IL-6 and its downstream molecule, phosphorylated-STAT3, correlated with expression of DNMT1. Concurrent with our animal and in-vitro studies, we analyzed 80 benign human prostate samples from TURPs and found that 24/80 (30%) of did not express the SRD5A2 protein with concurrent methylation of SRD5A2 promoter, suggesting that methylation and absence of of SRD5A2 protein expression are closely linked (p=0.0006, Fisher's exact test). This data suggests that expression of SRD5A2 is highly variable in human adult prostate tissue. CONCLUSIONS: Expression of SRD5A2 in aging prostates is a dynamic process that is regulated by methylation of SRD5A2 promoter. DNMT1 regulates the methylation and expression of SRD5A2. Dynamic and variable expression of SRD5A2 has implications for management of BPH and chemopreventive strategies for prostate cancer. Citation Format: Ge Rongbin, Zongwei Wang, Chin-Lee Wu, Shahin Tabatabaei, Aria Olumi. Dynamic expression of 5-alpha reductase 2 in aging prostate is regulated by DNA methyltransferase 1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2313. doi:10.1158/1538-7445.AM2014-2313

MP31-03 ALTERED HOXB13 FUNCTION IN HUMAN PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research II1 Apr 2014MP31-03 ALTERED HOXB13 FUNCTION IN HUMAN PROSTATE CANCER Shuangling Chen, Dorhyun Johng, Charles Ewing, and William Isaacs Shuangling ChenShuangling Chen More articles by this author , Dorhyun JohngDorhyun Johng More articles by this author , Charles EwingCharles Ewing More articles by this author , and William IsaacsWilliam Isaacs More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.912AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Our group, in collaboration with Dr. Kathleen Cooney’s lab at University of Michigan, recently identified a rare but recurrent germ line mutation (G84E) in HOXB13 as a high penetrance prostate cancer susceptibility allele (Ewing et al, NEJM 366:141). HOX genes are a family of homeodomain-containing transcription factors that play key roles in embryonic development, especially in the patterning of the anterior to posterior axis. HOX13 paralogs are particularly important in the development of the prostate gland in the mouse, but the function of HOXB13 in the adult human prostate and in prostate cancer development is unknown. To assess the role of wild-type (wt) HOXB13 in prostate biology and how this role might be altered by gene mutation to promote prostate carcinogenesis, we have initiated efforts to identify and characterize protein binding partners of HOXB13 in cell line models of prostate cancer, as well as to identify genes whose expression is modulated by varying expression levels of wt and mutant HOXB13. METHODS Mutant and wt HOXB13 were expressed by transduction in LNCaP cells. Endogenous HOXB13 was knocked down by siRNA targeting the 3’ UTR of the gene. Cells were treated with 10nM R1881 for 24 hours followed by gene expression microarray analysis. Gene Set Enrichment Analysis was performed to compare gene pathways differentially regulated by HOXB13 G84E. Co-immunoprecipitation (Co-IP) was performed to detect the interactions between AR, FOXA1, PBX2, PREP1, and HOXB13. RESULTS Cluster analysis of microarray gene expression data showed that over 250 genes were differentially regulated by HOXB13 G84E, including both androgen-dependent and -independent genes. Compared to wt HOXB13, G84E significantly down-regulated steroid metabolism pathway genes and up-regulated genes involved in polyamine metabolism. Co-IP results revealed that HOXB13 interacts with AR as previously demonstrated, and we identified the pioneer transcription factor FOXA1, and HOX cofactors, PBX2 and PREP1 as novel binding partners of HOXB13, at least in androgen responsive prostate cancer cell lines. CONCLUSIONS Differential expression of genes involved in steroid and polyamine metabolizing pathways might explain the ability of HOXB13 G84E to promote prostate carcinogenesis. Co-IP experiments demonstrate novel complex formation between HOXB13 and AR, FOXA1, PBX1, and PREP1. Further work is necessary to determine if alterations in this complex formation as a result of HOXB13 mutations are responsible for the altered gene expression patterns observed. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e323-e324 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Shuangling Chen More articles by this author Dorhyun Johng More articles by this author Charles Ewing More articles by this author William Isaacs More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Importance of HOX genes in normal prostate gland formation, prostate cancer development and its early detection

The aims of this paper were to review the published literature on the role of HOX genes in the development of the normal prostate gland and in prostate cancer and to discuss the potential role of the HOX family member, Engrailed-2 (EN2), as a diagnostic test of PCa. Hox genes were first described in the fruit fly Drosphila melanogaster, where they specify the body plan and control the formation of body segments. They belong to a family of homeodomain-containing transcription factors that determine cell and tissue identity during normal embryonic development. They have been shown to be re-expressed by several different types of cancers. Studies have shown that different Hox genes are responsible for the development of the separate lobes of the prostate gland, the seminal vesicles and the epididymis. All HOX13 paralogues are expressed in the adult human prostate, suggesting the possibility of similarities between the function and expression of HOX genes within urological structures at similar anterior-posterior positions. The oncogenic and tumour suppressor signalling pathways associated with PCa converge on the HOX gene network, which ultimately controls gene expression, affecting tumour formation and metastatic progression. The Engrailed genes (EN1 and EN2) from the HOX gene family show a very high degree of functional conservation during embryonic development. Urinary EN2 is being investigated as a potential diagnostic marker of early PCa. It is secreted into the urine by PCa cells but not by normal prostatic tissue. A recent study has shown an association between urinary EN2 levels and cancer volume in radical prostatectomy specimens. The ability to predict tumour volume could inform the treatment decision-making process for patients with localized PCa choosing between active surveillance and radical treatment options.

Growth kinetics of CD133‐positive prostate cancer cells

In the adult human prostate CD133 expression is thought to mark rare prostate epithelial stem cells and malignant tumor stem/initiating cells. Such putative stem cell populations are thought to proliferate slowly, but possess unlimited proliferative potential. Based on this, we hypothesized that CD133(pos) prostate cancer cells proliferate slower than CD133(neg) cells. Human prostate cancer cell lines were analyzed for CD133 expression and DNA content using flow cytometry. Rates of cell division and DNA synthesis were determined using CFSE cell tracing and BrdU uptake, respectively. Changes in cell cycle distribution and the percentage of CD133(pos) cells were assayed under conditions of different cell density and AR-pathway modulation. Lastly, we over-expressed lentiviral CD133 to measure whether CD133 regulates the cell cycle. The cell cycle distribution differs between CD133(pos) and CD133(neg) cells in all three human prostate cancer cell lines studied. CD133(pos) cells have a greater proportion of cells in G2 and proliferate faster than CD133(neg) cells. High cell density increases the percentage of CD133(pos) cells without changing CD133(pos) cell cycle progression. Treatment with the AR agonist R1881, or the anti-androgen MDV3100, significantly changed the percentage and proliferation of CD133(pos) cells. Finally, ectopic over-expression of CD133 had no effect on cell cycle progression. Contrary to our hypothesis, we demonstrate that CD133(pos) cells proliferate faster than CD133(neg) cells. This association of CD133 expression with increased cell proliferation is not directly mediated by CD133, suggesting that surface CD133 is a downstream target gene of an undefined pathway controlling cell proliferation.

Steroidogenic potential of adult mouse and human prostate and penis

The major steroidogenic tissues in the male are well known. Expression of p450scc, 3β-HSD and StAR are well established in the adrenal gland and in the testis. Steroidogenic acute regulatory protein (StAR) represents the first and rate-limiting step for steroid biosynthesis leading to androgen production followed by the metabolism of cholesterol to pregnenolone by the cytochrome p450 side chain enzyme (p450scc). We tested the hypothesis that organs not usually considered to be steroidogenic, exhibit steroidogenic potential.

Reduced levels of 5‐α reductase 2 in adult prostate tissue and implications for BPH therapy

5-α reductase 2 (5-AR 2) is a key enzyme that is responsible of proper development of prostate tissue. Inhibition of 5-AR 2 has proven to be efficacious for management of urinary symptoms secondary benign prostatic hyperplasia (BPH). However, some patients are resistant to the therapeutic effects of 5-AR 2 inhibitor. We wished to determine why some benign non-cancerous adult human prostates do not express 5-AR 2, and hypothesized that methylation of 5-AR 2 promoter region correlated with low expression of 5-AR 2 protein. The transition zone of 42 human prostate tissues after radical prostatectomy was used for evaluation. Initially, 21 paraffin embedded samples were used to assess immunoreactivity to 5-AR 2 antibody in non-cancerous BPH samples. In the next 21 samples, fresh frozen prostate transition zone samples without cancer were assessed for immunoreactivity and methylation of the 5-AR 2 promoter using methyl-specific PCR. We show that 6/21 (29%) of benign human prostate samples did not express the 5-AR 2 protein. Moreover, the promoter region of 5-AR 2 contains a CpG island that is methylated in benign prostate epithelial cells in culture and also in 39% (7/18) human prostate tissues. We show a strong correlation between methylation of the 5-AR 2 promoter region and absence of 5-AR 2 protein expression (P = 0.0025, Fisher's exact test). Methylation of 5-AR 2 promoter may account for low or absent expression of 5-AR 2 in some human adult prostate tissues.