Normal Prostate Epithelium Research Articles

Estrogen-initiated transformation of prostate epithelium derived from normal human prostate stem-progenitor cells.

The present study sought to determine whether estrogens with testosterone support are sufficient to transform the normal human prostate epithelium and promote progression to invasive adenocarcinoma using a novel chimeric prostate model. Adult prostate stem/early progenitor cells were isolated from normal human prostates through prostasphere formation in three-dimensional culture. The stem/early progenitor cell status and clonality of prostasphere cells was confirmed by immunocytochemistry and Hoechst staining. Normal prostate progenitor cells were found to express estrogen receptor α, estrogen receptor β, and G protein-coupled receptor 30 mRNA and protein and were responsive to 1 nm estradiol-17β with increased numbers and prostasphere size, implicating them as direct estrogen targets. Recombinants of human prostate progenitor cells with rat urogenital sinus mesenchyme formed chimeric prostate tissue in vivo under the renal capsule of nude mice. Cytodifferentiation of human prostate progenitor cells in chimeric tissues was confirmed by immunohistochemistry using epithelial cell markers (p63, cytokeratin 8/18, and androgen receptor), whereas human origin and functional differentiation were confirmed by expression of human nuclear antigen and prostate-specific antigen, respectively. Once mature tissues formed, the hosts were exposed to elevated testosterone and estradiol-17β for 1-4 months, and prostate pathology was longitudinally monitored. Induction of prostate cancer in the human stem/progenitor cell-generated prostatic tissue was observed over time, progressing from normal histology to epithelial hyperplasia, prostate intraepithelial neoplasia, and prostate cancer with local renal invasion. These findings provide the first direct evidence that human prostate progenitor cells are estrogen targets and that estradiol in an androgen-supported milieu is a carcinogen for human prostate epithelium.

Identification of a small-molecule inhibitor of Stat5a/b through structure-based screen for therapy development for prostate cancer.

17 Background: There are no effective treatments for metastatic or castration resistant prostate cancer. We have shown that transcription factor Stat5a/b is constitutively active in high-grade prostate cancer, but not in normal human prostate epithelium. Stat5a/b is active in 95% of clinical castration resistant prostate cancers, and the expression of active Stat5a/b in primary prostate cancer predicts early disease recurrence. Stat5a/b is critical for the viability of prostate cancer cells in vitro and for growth of prostate xenograft tumors in nude mice. Stat5a/b synergizes with androgen receptor (AR) and Stat5a/b promotes metastatic behavior of human prostate cancer cells in vitro and in vivo. Here, we hypothesize that Stat5a/b is a molecular target for rational drug design for prostate cancer. Methods: We identified a small- molecule inhibitor of Stat5a/b dimerization by structure-based virtual screen from a database of 30 million chemical structures. The efficacy of the Stat5a/b inhibitor was determined by reporter gene assays, dimerization by co-immunoprecipitations, nuclear translocation by cytochemistry and binding to DNA by EMSA. Cell viability was analyzed by MTT assay. Results: The novel Stat5a/b inhibitor IST5-002 inhibited transcriptional activity of Stat5a/b at IC50 of 1.5 μ M for Stat5a and 3.5 μ M for Stat5b, but not of Stat3 in prostate cancer cells. IST5-002 inhibited dimerization, nuclear translocation, and binding of Stat5a/b to the Stat5 DNA consensus sequence. Furthermore, IST5-002 inhibited expression of Stat5a/b target gene cyclin D1, and induced massive apoptosis of DU145, CWR22Rv1 and LNCaP human prostate cancer cells. IST5-002 blocked prostate cancer xenograft tumor growth in nude mice and induced death in clinical prostate cancers ex vivo in 3D organ cultures. Conclusions: We have identified a small molecule Stat5a/b inhibitor IST5-002 for therapy development for prostate cancer. Future work will focus on chemical modifications of IST5-002 to achieve IC50 below 1 μ M and oral administration. No significant financial relationships to disclose.

SMAD4-dependent barrier constrains prostate cancer growth and metastatic progression

Effective clinical management of prostate cancer (PCA) has been challenged by significant intratumoural heterogeneity on the genomic and pathological levels and limited understanding of the genetic elements governing disease progression. Here, we exploited the experimental merits of the mouse to test the hypothesis that pathways constraining progression might be activated in indolent Pten-null mouse prostate tumours and that inactivation of such progression barriers in mice would engender a metastasis-prone condition. Comparative transcriptomic and canonical pathway analyses, followed by biochemical confirmation, of normal prostate epithelium versus poorly progressive Pten-null prostate cancers revealed robust activation of the TGFβ/BMP-SMAD4 signalling axis. The functional relevance of SMAD4 was further supported by emergence of invasive, metastatic and lethal prostate cancers with 100% penetrance upon genetic deletion of Smad4 in the Pten-null mouse prostate. Pathological and molecular analysis as well as transcriptomic knowledge-based pathway profiling of emerging tumours identified cell proliferation and invasion as two cardinal tumour biological features in the metastatic Smad4/Pten-null PCA model. Follow-on pathological and functional assessment confirmed cyclin D1 and SPP1 as key mediators of these biological processes, which together with PTEN and SMAD4, form a four-gene signature that is prognostic of prostate-specific antigen (PSA) biochemical recurrence and lethal metastasis in human PCA. This model-informed progression analysis, together with genetic, functional and translational studies, establishes SMAD4 as a key regulator of PCA progression in mice and humans.

Abstract B65: Cyclin-dependent kinase inhibitor p21 is a crucial molecular target for the potential antitumor efficacy of the dietary isothiocyanate erucin against prostate cancer

Abstract It is becoming increasingly clear that many dietary agents, such as isothiocyanates (ITCs) from cruciferous vegetables, can retard or prevent the process of prostate carcinogenesis by multiple mechanisms [1, 2]. Erucin (ER) is a dietary isothiocyanate present in cruciferous vegetables, which has been recently considered a new promising cancer chemopreventive phytochemical [2]. In this study, the potential protective activity of ER against prostate cancer was investigated using two different human prostatic cell lines, normal prostate epithelium (PNT1A) and prostate adenocarcinoma cells (PC3), in order to analyze its effects on molecular pathways involved in cell growth regulation, such as the cyclin-dependent kinase (CDKs) inhibitor p21WAF1/CIP1 (p21) pathway [3]. We have shown for the first time that the isothiocyanate ER may up-regulate significantly p21 protein expression in a dose-dependent manner to inhibit the proliferation of human adenocarcinoma prostate PC3 cells. Compared to the structurally related sulforaphane (SF), a well-studied broccoli-derived ITC, the isothiocyanate ER showed lower potency in inhibiting proliferation of PC3 cells, as well as in modulating p21 protein expression. Several studies have showed that the tumor suppressor protein p53 plays a key role in cellular response to DNA damage leading to apoptosis in case of unrepaired DNA damage, or indirectly to the block of cell cycle progression by transactivating p21 [4]. Because PC3 cells do not express functional p53, our data suggest that the up-regulation of p21 by ER occurs through a p53-independent pathway. Moreover, our results showed that ER and SF did not affect p21 protein levels in normal prostate epithelium PNT1A cells at all concentrations tested (0-25 µM). These preliminary data demonstrate that the above structurally related ITCs exert a specific action on oncogenic signaling pathways, such as p53/p21 pathways, that could explain the stronger cytotoxicity observed in cancer cells compared to non malignant cells. Although these dietary bioactive compounds have exhibited selectivity for oncogenically transformed cell lines, ITCs selectivity is an area of great interest that warrants further investigation.

Differential expression of anterior gradient gene AGR2 in prostate cancer

BackgroundThe protein AGR2 is a putative member of the protein disulfide isomerase family and was first identified as a homolog of the Xenopus laevis gene XAG-2. AGR2 has been implicated in a number of human cancers. In particular, AGR2 has previously been found to be one of several genes that encode secreted proteins showing increased expression in prostate cancer cells compared to normal prostatic epithelium.MethodsGene expression levels of AGR2 were examined in prostate cancer cells by microarray analysis. We further examined the relationship of AGR2 protein expression to histopathology and prostate cancer outcome on a population basis using tissue microarray technology.ResultsAt the RNA and protein level, there was an increase in AGR2 expression in adenocarcinoma of the prostate compared to morphologically normal prostatic glandular epithelium. Using a tissue microarray, this enhanced AGR2 expression was seen as early as premalignant PIN lesions. Interestingly, within adenocarcinoma samples, there was a slight trend toward lower levels of AGR2 with increasing Gleason score. Consistent with this, relatively lower levels of AGR2 were highly predictive of disease recurrence in patients who had originally presented with high-stage primary prostate cancer (P = 0.009).ConclusionsWe have shown for the first time that despite an increase in AGR2 expression in prostate cancer compared to non-malignant cells, relatively lower levels of AGR2 are highly predictive of disease recurrence following radical prostatectomy.

Enhanced noscapine delivery using uPAR-targeted optical-MR imaging trackable nanoparticles for prostate cancer therapy

The tubulin-binding anticancer activity of noscapine, an orally available plant-derived anti-tussive alkaloid, has been recently identified. Noscapine inhibits tumor growth in nude mice bearing human xenografts of hematopoietic, breast, lung, ovarian, brain and prostate origin. Despite its nontoxic attributes, significant elimination of the disease has not been achieved, perhaps since the bioavailability of noscapine to tumors saturates at an oral dose of 300 mg/kg body weight. To enable the selective and specific delivery of noscapine to prostate cancer cells, we have engineered a multifunctional nanoscale delivery vehicle that takes advantage of urokinase plasminogen activator receptor (uPAR) overexpression in prostate cancer compared to normal prostate epithelia and can be tracked by magnetic resonance imaging (MRI) and near-infrared (NIR) imaging. Specifically, we employed the human-type 135 amino-acid amino-terminal fragment (hATF) of urokinase plasminogen activator (uPA), a high-affinity natural ligand for uPAR. Noscapine (Nos) was efficiently adsorbed onto the amphiphilic polymer coating of uPAR-targeted nanoparticles (NPs). Nos-loaded NPs were uniformly compact-sized, stable at physiological pH and efficiently released the drug at pH 4 to 5 within a span of 4 h. Our results demonstrate that these uPAR-targeted NPs were capable of binding to the receptor and were internalized by PC-3 cells. uPAR-targeted Nos-loaded NPs enhanced intracellular noscapine accumulation as evident by the ~ 6-fold stronger inhibitory effect on PC-3 growth compared to free noscapine. In addition, Nos-loaded iron oxide NPs maintained their T2 MRI contrast effect upon internalization into tumor cells owing to their significant susceptibility effect in cells. Thus, our data provide compelling evidence that these optically and magnetic resonance imaging (MRI)-trackable uPAR-targeted NPs may offer a great potential for image-directed targeted delivery of noscapine for the management of prostate cancer.

Expression pattern of REIC/Dkk-3 in various cell types and the implications of the soluble form in prostatic acinar development

The tumor suppressor REIC/Dkk-3 is a secretory protein which was originally identified to be downregulated in human immortalized cells. In the present study, we investigated the expression pattern of REIC/Dkk-3 in various cell types to characterize its physiological functions. We first examined the expression level of REIC/Dkk-3 in a broad range of cancer cell types and confirmed that it was significantly downregulated in all of the cell types. We also examined the tissue distribution pattern in a variety of normal mouse organs. Ubiquitous REIC/Dkk-3 protein expression was observed in the organs. The expression was abundant in the liver, heart and brain tissue, but was absent in the spleen and peripheral blood mononuclear cells. The immunohistochemical analyses revealed that the subcellular localization of REIC/Dkk-3 had a punctate pattern around the nucleus, indicating its association with secretory vesicles. In cancer cells stably transfected with REIC/Dkk-3, the protein was predominantly localized to the endoplasmic reticulum (ER) under observation with confocal microscopy. Because REIC/Dkk-3 was found to be abundantly expressed in the acinar epithelial cells of the mouse prostate, we analyzed the effects of recombinant REIC/Dkk-3 protein on the acinar morphogenesis of RWPE-1 cells, which are derived from human normal prostate epithelium. Statistically significant acinar growth was observed in the culture condition with 10 µg/ml REIC/Dkk-3 protein, implicating the soluble form in prostatic acinar development. Current results suggest that REIC/Dkk-3 may play a role in regulating the morphological process of normal tissue architecture through an autocrine and/or paracrine manner.

Changes in cortical cytoskeletal and extracellular matrix gene expression in prostate cancer are related to oncogenic ERG deregulation

BackgroundThe cortical cytoskeleton network connects the actin cytoskeleton to various membrane proteins, influencing cell adhesion, polarity, migration and response to extracellular signals. Previous studies have suggested changes in the expression of specific components in prostate cancer, especially of 4.1 proteins (encoded by EPB41 genes) which form nodes in this network.MethodsExpression of EPB41L1, EPB41L2, EPB41L3 (protein: 4.1B), EPB41L4B (EHM2), EPB41L5, EPB49 (dematin), VIL2 (ezrin), and DLG1 (summarized as „cortical cytoskeleton" genes) as well as ERG was measured by quantitative RT-PCR in a well-characterized set of 45 M0 prostate adenocarcinoma and 13 benign tissues. Hypermethylation of EPB41L3 and GSTP1 was compared in 93 cancer tissues by methylation-specific PCR. Expression of 4.1B was further studied by immunohistochemistry.ResultsEPB41L1 and EPB41L3 were significantly downregulated and EPB41L4B was upregulated in cancer tissues. Low EPB41L1 or high EPB41L4B expression were associated with earlier biochemical recurrence. None of the other cortical cytoskeleton genes displayed expression changes, in particular EPB49 and VIL2, despite hints from previous studies. EPB41L3 downregulation was significantly associated with hypermethylation of its promoter and strongly correlated with GSTP1 hypermethylation. Protein 4.1B was detected most strongly in the basal cells of normal prostate epithelia. Its expression in carcinoma cells was similar to the weaker one in normal luminal cells. EPB41L3 downregulation and EPB41L4B upregulation were essentially restricted to the 22 cases with ERG overexpression. Expression changes in EPB41L3 and EPB41L4B closely paralleled those previously observed for the extracellular matrix genes FBLN1 and SPOCK1, respectively.ConclusionsSpecific changes in the cortical cytoskeleton were observed during prostate cancer progression. They parallel changes in the expression of extracellular matrix components and all together appear to be associated with oncogenic ERG overexpression. We hypothesize that these alterations may contribute to the increased invasivity conferred to prostate cancer cells by ERG deregulation.

SENP1 Induces Prostatic Intraepithelial Neoplasia through Multiple Mechanisms

SUMOylation has been shown to modulate DNA replication/repair, cell cycle progression, signal transduction, and the hypoxic response. SUMO (small ubiquitin-like modifier)-specific proteases regulate SUMOylation, but how changes in the expression of these proteases contribute to physiological and/or pathophysiological events remains undefined. Here, we show that SENP1 (sentrin/SUMO-specific protease 1) is highly expressed in human prostate cancer specimens and correlates with hypoxia-inducing factor 1alpha (HIF1alpha) expression. Mechanistic studies in a mouse model indicate that androgen-driven expression of murine SENP1 leads to HIF1alpha stabilization, enhanced vascular endothelial growth factor production, and angiogenesis. Further pathological assessment of the mouse indicates that SENP1 overexpression induces transformation of the normal prostate gland and gradually facilitates the onset of high-grade prostatic intraepithelial neoplasia. Consistent with cell culture studies, SENP1 enhances prostate epithelial cell proliferation via modulating the androgen receptor and cyclin D(1). These results demonstrate that deSUMOylation plays a critical role in prostate pathogenesis through induction of HIF1alpha-dependent angiogenesis and enhanced cell proliferation.

Androgen-induced TOP2B-mediated double-strand breaks and prostate cancer gene rearrangements

DNA double strand breaks (DSB) can lead to development of genomic rearrangements, which are hallmarks of cancer. TMPRSS2-ERG gene fusions in prostate cancer (PCa) are among the most common genomic rearrangements observed in human cancer. We show that androgen signaling promotes co-recruitment of androgen receptor (AR) and topoisomerase II beta (TOP2B) to sites of TMPRSS2-ERG genomic breakpoints, triggering recombinogenic TOP2B-mediated DSB. Furthermore, androgen stimulation resulted in de novo production of TMPRSS2-ERG fusion transcripts in a process requiring TOP2B and components of DSB repair machinery. Finally, unlike normal prostate epithelium, prostatic intraepithelial neoplasia (PIN) cells showed strong co-expression of AR and TOP2B. These findings implicate androgen-induced TOP2B-mediated DSB in generating TMPRSS2-ERG rearrangements.

SUMO Losing Balance: SUMO Proteases Disrupt SUMO Homeostasis to Facilitate Cancer Development and Progression

Small ubiquitin-like modifiers (SUMO) conjugation to cellular proteins is a reversible posttranslational modification that mediates the protein's function, subcellular localization, and/or expression. The SUMO proteases (SENP) deconjugate modified proteins and thus are critical for maintaining the level of SUMOylated and un-SUMOylated substrates required for normal physiology. Altered expression of SENPs is observed in several carcinomas. This review focuses on how the change in SENP levels disturbs SUMO homeostasis and contributes to cancer development and progression. We reported that one member of the SENP family, SENP1 can transform normal prostate epithelia to a dysplasic state and directly modulate several oncogenic pathways in prostate cells, including AR, c-Jun, and Cyclin D1. Assessment of tissue from human prostate cancer patients indicates elevated mRNA levels of SENP1 and the SUMO2/3 deconjugating enzyme, SENP3. The induction of SENP3 in cancer cells initiates the angiogenic pathway; specifically SENP3 regulates the transcriptional activity of hypoxia-inducible factor 1α (HIF1α) via deSUMOylation of the co-regulatory protein p300. Unlike prostate cancer, enhanced SUMOylation is favored with onset of breast cancer and correlated with the reduced SENP6 mRNA levels found in several breast cancer tissue arrays. Preventing enhanced SUMO conjugation of cellular substrates in breast cancer cells reduces tumorigenesis. Hence, distortion of SUMO equilibrium contributes to both the initiation and progression of cancer, specifically in prostate and breast cancers. The deSUMOylation machinery may be key to restoring balance to the SUMO system and hence serve as ideal targets for therapeutic agents.

836 Effects of irradiation on viability, growth, metastatic properties and expression of Eph receptors and their ephrin ligands in human melanoma cells

The family of Eph and ephrin receptors is involved in a variety of functions in normal cells, and the alterations in their expression profiles have been observed in several cancers. We have compared the transcripts for Eph receptors and ephrin ligands in cell lines established from normal prostate epithelium and several carcinoma cell lines isolated from prostate tumors of varying degree of metastasis. These cell lines included NPTX, CTPX, LNCaP, DU145, PC-3, and PC-3ML. The cell lines displayed characteristic pattern of expression for specific Eph receptors and ephrin ligands, thus allowing identification of Eph receptor signatures for a particular cell line. The sensitivity of these transcripts to genome methylation is also investigated by treating the cells with 5-aza-2′-deoxycytidine. The comparison of expression profiles revealed that normal prostate and primary prostate tumor cell lines differ in the expression of EphA3, EphB3, and ephrin A3 that are over-expressed in normal prostate. Furthermore, the transcript levels for EphA1 decrease progressively from normal prostate to primary prostate tumor cell line and metastatic tumor cells. A converse relationship was observed for ephrin B2. The treatment of cells with 5-aza-2′-deoxycytidine revealed the sensitivity of EphA3, EphA10, EphB3, and EphB6 to methylation status of genomic DNA. The utility of methylation specific PCR to identify prostate tumor cells and the importance of specific Eph receptors and ephrin ligands in initiation and progression of prostate tumor are discussed.

The chromatin remodeling factor SRCAP modulates expression of prostate specific antigen and cellular proliferation in prostate cancer cells

The SNF2-related CBP activator protein (SRCAP) serves as a coactivator for several nuclear receptors including the androgen receptor (AR). SRCAP is an ATPase that is the core subunit of a large multiprotein complex and was shown to incorporate the histone variant H2A.Z into nucleosomes. In this report, we demonstrate that SRCAP is expressed in the epithelium of normal prostate and in prostate carcinoma cells, and is associated with AR in the nucleus. Using transient transfection assays we demonstrate that SRCAP activates hormone-dependent transcription of the androgen responsive, prostate specific antigen (PSA)-Luciferase reporter gene in human prostate cells. The in vivo occupancy of SRCAP at the endogenous PSA promoter is demonstrated using chromatin immunoprecipitation assays. ShRNA mediated knockdown of SRCAP resulted in decreased H2A.Z binding at the enhancer region of the PSA promoter and decreased expression of PSA in prostate cancer cells. Furthermore, inhibition of SRCAP expression significantly inhibited androgen dependent prostate cancer cell growth. These data identify SRCAP as a physiologically relevant mediator of PSA expression, and demonstrate that SRCAP plays a role in prostate cancer cell proliferation.

The distribution of PAX-2 immunoreactivity in the prostate gland, seminal vesicle, and ejaculatory duct: comparison with prostatic adenocarcinoma and discussion of prostatic zonal embryogenesis

PAX-2 is a homeogene strongly expressed during development of the genitourinary tract, including the kidney and both wolffian- and müllerian-derived tissues. Expression of PAX-2 by immunohistochemistry has been studied mainly in renal epithelial neoplasms with little attention to the lower male genitourinary tract. We studied PAX-2 expression in epithelium of normal seminal vesicle, normal ejaculatory duct, normal prostatic secretory epithelium, and prostatic adenocarcinoma to define its immunoreactivity pattern throughout the prostate gland and to evaluate its potential diagnostic role in the discrimination of seminal vesicle/ejaculatory duct epithelium from prostatic adenocarcinoma. In addition, given that PAX-2 is highly expressed in tissues of wolffian duct embryologic origin, we also sought to confirm the divergent embryogenesis of the central zone, seminal vesicle, and ejaculatory duct from other regions of the prostate. Prostatectomy specimens from 12 patients were reviewed to identify blocks containing seminal vesicle, ejaculatory duct, periurethral glands, benign prostatic glands, and prostatic acinar adenocarcinoma. A total of 35 blocks from the 12 patients were evaluated. In addition, 2 tissue microarrays representing 15 additional seminal vesicles and 45 prostatic adenocarcinomas, 7 whole sections from prostatic adenocarcinomas of the central zone, and 5 core needle biopsies of seminal vesicle were also evaluated with anti-PAX-2 antibody. In the 12 radical prostatectomy whole sections, nuclear reactivity for PAX-2 was identified in 12 (100%) of 12 of the seminal vesicle epithelium, 9 (90%) of 10 of the ejaculatory duct epithelium, 0 of 12 of the prostatic adenocarcinoma, and 0 of 6 of the high-grade prostatic intraepithelial neoplasia. All 20 total additional seminal vesicles were positive for PAX-2 in the tissue microarray and biopsies; and all 52 additional prostatic adenocarcinomas were negative, including 7 of central zone origin. The staining intensity and percentage of immunoreactive cells in seminal vesicle were both 3+ in all cases. Although the ejaculatory ducts also showed diffuse staining, their staining intensity was less (2+) than that in the seminal vesicles, particularly in the ejaculatory ducts in the periurethral area (1-2+intensity). The smaller glands surrounding the main seminal vesicle duct also showed less intense staining than the luminal cells of the main duct. Of the 19 total cases with evaluable central zone glands, 2 (10.5%) had focal nuclear reactivity in normal, benign prostatic secretory cells. All other benign prostatic secretory epithelia from the peripheral and transition zones were negative for PAX-2. In conclusion, nuclear PAX-2 immunoreactivity is typical in epithelium of the seminal vesicle and ejaculatory duct; but the intensity of staining is less in the ejaculatory duct. No reactivity for PAX-2 was seen in prostatic adenocarcinoma or high-grade prostatic intraepithelial neoplasia. PAX-2 has diagnostic utility as a positive immunohistochemical marker of seminal vesicle and ejaculatory duct epithelium. In addition, these data add further support to the proposed embryogenesis of the prostatic central zone, seminal vesicle, and ejaculatory ducts from the wolffian system.

Abstract 345: Prostaglandin receptors differentially regulate prostate cancer growth: A novel treatment target

Abstract INTRODUCTION AND OBJECTIVE: Prostaglandin E2 (PGE2) acts through a series of distinct prostanoid receptors, EPR 1-4. Since PGE2 is implicated in prostate cancer (CaP) tumorigenesis and metastasis, we hypothesized that EP receptor expression may influence CaP growth. METHODS: 27 consecutive radical prostatectomy specimens were assessed by immunohistochemistry (IHC) for EPR expression and compared with normal human prostate epithelium (NP) from the same specimen as control. As a corollary, the expression of EP receptors in both normal (PrEC, PWR1E and PZ-HPV7) and CaP cells (LNCaP, PC3 and CA-HPV10) were assessed by RT-PCR and Western blot. The functional significance of EPR was assessed by growth of malignant and benign cell lines in vitro after siRNA EPR knockdown. All studies were approved by the local institutional review board (IRB) and all in vitro experiments performed a minimum of 3 times. RESULTS: EP1, EP2, EP3 and EP4 receptors were detected by IHC in all NP clinical specimens. Compared with NP, EP4 and EP2 were over-expressed in 74% (20/27) and 63% (17/27) of CaP specimens, respectively; in contrast, EP3 expression was reduced in 93% (25/27) clinical samples (Figure). EP1 showed no specific differential expression pattern. Western blot showed increased EP4 but reduced EP3 in CaP lines (CA-HPV10, LNCaP and PC3) compared with NP lines; EP2 and EP4 knockdown significantly suppressed whereas EP3 knockdown stimulated in vitro growth of CA-HPV10 vs PZ-HPV7 cells. Expression of Runx 2 and MMP9, (known to contribute to CaP metastatic potential) was also reduced after EP2 and EP4 knockdown in CaP cells. CONCLUSION: We observed EP4 and EP2 over-expression and reduced EP3 expression in 27 consecutive resected CaP specimens and in 6 representative cell lines. These observations were corroborated by the effects of EP4, EP2 and EP3 knockdown on tumor cell growth in vitro. These data suggest that EP receptor subtypes may regulate CaP growth which may represent a novel therapeutic approach. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 345.

559 HUMAN HETEROCHROMATIN PROTEIN 1 ISOFORM HP1βASSOCIATED WITH METHYLATED LYS9 OF HISTONE H3 PROMOTES PROSTATE CANCER CELL GROWTH THROUGH ANDROGEN RECEPTOR ACTIVATION

You have accessJournal of UrologyProstate Cancer: Basic Research IV1 Apr 2010559 HUMAN HETEROCHROMATIN PROTEIN 1 ISOFORM HP1βASSOCIATED WITH METHYLATED LYS9 OF HISTONE H3 PROMOTES PROSTATE CANCER CELL GROWTH THROUGH ANDROGEN RECEPTOR ACTIVATION Masaki Shiota, YooHuang Song, Akira Yokomizo, Yasuhiro Tada, Junichi Inokuchi, Daisuke Masubuchi, Masatoshi Eto, Naohiro Fujimoto, Narihito Seki, and Seiji Naito Masaki ShiotaMasaki Shiota Fukuoka, Japan More articles by this author , YooHuang SongYooHuang Song Fukuoka, Japan More articles by this author , Akira YokomizoAkira Yokomizo Fukuoka, Japan More articles by this author , Yasuhiro TadaYasuhiro Tada Fukuoka, Japan More articles by this author , Junichi InokuchiJunichi Inokuchi Fukuoka, Japan More articles by this author , Daisuke MasubuchiDaisuke Masubuchi Fukuoka, Japan More articles by this author , Masatoshi EtoMasatoshi Eto Kumamoto, Japan More articles by this author , Naohiro FujimotoNaohiro Fujimoto Kitakyushu, Japan More articles by this author , Narihito SekiNarihito Seki Fukuoka, Japan More articles by this author , and Seiji NaitoSeiji Naito Fukuoka, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.779AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES There are currently few successful therapies for castration-resistant prostate cancer (CRPC). CRPC results from augmented androgen/androgen receptor (AR) signaling pathway, which could be enhanced by AR cofactors. Recently, epigenetics have been known to play the important role in gene expression. Then, we searched new AR cofactor from the stand of epigenetics and evaluated the function of newly-identified AR cofactor. In addition, the possibility as therapeutic target was evaluated. METHODS Human prostate cancer PC-3, LNCaP cells and castration-resistant LNCaP derivatives which were established by us and named as CxR cells were used. Using these cells, we searched the AR cofactor relating to epigenetics by co-immunoprecipitation assay. Based on the identified AR cofactor, we performed luciferase reporter assay using PSA reporter plasmid to examine the role as AR cofactor. Further, we conducted chromatin-immunoprecipitation assay to evaluate AR binding ability to PSA promoter. In addition, immunohistochemistry was performed using prostate cancer tissues. Last, we evaluated the impact as a therapeutic target based on the identified AR cofactor by cell proliferation assay and flow cytometry. RESULTS We identified new AR co-factor, heterochromatin protein 1 isoform, HP1β. HP1β was shown to upregulate the PSA transcription via augmentation of binding to androgen-responsive elements (Fig. A). In addition, CxR cells expressed high levels of HP1β and AR protein. Immunohistochemistry of prostate cancer revealed that HP1β expression and tri-methylated lysine 9 of histone H3 level was elevated in cancer compared with normal prostate epithelium and HP1β expression correlated with Gleason score (Fig. B). Silencing of HP1β suppressed cell growth of LNCaP and CxR cells by inducing cell-cycle arrest (Fig. C). CONCLUSIONS This study indicated that HP1β is involved in proliferation of AR-expressing prostate cancer cells and progression to CRPC as an AR coactivator. Modulation of HP1β expression or function might be a useful strategy for developing novel therapeutics for prostate cancer, even in CRPC. © 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e219-e220 Peer Review Report Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Masaki Shiota Fukuoka, Japan More articles by this author YooHuang Song Fukuoka, Japan More articles by this author Akira Yokomizo Fukuoka, Japan More articles by this author Yasuhiro Tada Fukuoka, Japan More articles by this author Junichi Inokuchi Fukuoka, Japan More articles by this author Daisuke Masubuchi Fukuoka, Japan More articles by this author Masatoshi Eto Kumamoto, Japan More articles by this author Naohiro Fujimoto Kitakyushu, Japan More articles by this author Narihito Seki Fukuoka, Japan More articles by this author Seiji Naito Fukuoka, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Targeting monoamine oxidase A in advanced prostate cancer

Inhibitors of monoamine oxidase A (MAOA), a mitochondrial enzyme that degrades neurotransmitters including serotonin and norepinephrine, are commonly used to treat neurological conditions including depression. Recently, we and others identified high expression of MAOA in normal basal prostatic epithelium and high-grade primary prostate cancer (PCa). In contrast, MAOA is low in normal secretory prostatic epithelium and low-grade PCa. An irreversible inhibitor of MAOA, clorgyline, induced secretory differentiation in primary cultures of normal basal epithelial cells and high-grade PCa. Furthermore, clorgyline inhibited several oncogenic pathways in PCa cells, suggesting clinical value of MAOA inhibitors as a pro-differentiation and anti-oncogenic therapy for high-risk PCa. Here, we extended our studies to a model of advanced PCa, VCaP cells, which were derived from castration-resistant metastatic PCa and express a high level of MAOA. Growth of VCaP cells in the presence or absence of clorgyline was evaluated in vitro and in vivo. Gene expression changes in response to clorgyline were determined by microarray and validated by quantitative real-time polymerase chain reaction. Treatment with clorgyline in vitro inhibited growth and altered the transcriptional pattern of VCaP cells in a manner consistent with the pro-differentiation and anti-oncogenic effects seen in treated primary PCa cells. Src, beta-catenin, and MAPK oncogenic pathways, implicated in androgen-independent growth and metastasis, were significantly downregulated. Clorgyline treatment of mice bearing VCaP xenografts slowed tumor growth and induced transcriptome changes similar to those noted in vitro. Our results support the possibility that anti-depressant drugs that target MAOA might find a new application in treating PCa.

Activating Mutation (V617F) in the Tyrosine Kinase JAK2 is Absent in Locally-Confined or Castration-Resistant Prostate Cancer

Background: Transcription factor Stat5a/b is highly critical for the viability of human prostate cancer cells in vitro and for prostate tumor growthin vivo. Stat5 is constitutively active in clinical prostate cancers but not in the normal human prostate epithelium. Moreover, Stat5a/b activation in prostate cancer is associated with high histological grade of prostate cancer. However, the molecular mechanisms underlying constitutive activation of Stat5a/b in prostate cancer are unclear. The receptor-associated tyrosine kinase Jak2 is a known key activator of Stat5a/b in prostate cancer cells in response to ligand stimulation. Recently, a single gain-of-function point mutation ofJAK2was described in myeloproliferative diseases leading to constitutive Jak2 kinase activity, subsequent Stat5a/b activation and involvement of V617F Jak2 in the pathogenesis of myeloproliferative disorders.Materials and Methods: We determined whetherJAK2undergoes the V617F activating mutation during clinical progression of human prostate cancer using a highly sensitive assay (amplification refractory mutation system) and a unique material of fresh specimens from organ-confined or castration-resistant prostate cancers.Results: TheJAK2V617F mutation was not found in any of the normal or malignant prostate samples analyzed in this study.Conclusions: Future work should focus on determining the molecular mechanisms other than V617F mutation of Jak2 resulting in continuous Stat5 activation in clinical prostate cancers.

Activating mutation (V617F) in the tyrosine kinase JAK2 is absent in locally-confined or castration-resistant prostate cancer.

Background: Transcription factor Stat5a/b is highly critical for the viability of human prostate cancer cells in vitro and for prostate tumor growth in vivo. Stat5 is constitutively active in clinical prostate cancers but not in the normal human prostate epithelium. Moreover, Stat5a/b activation in prostate cancer is associated with high histological grade of prostate cancer. However, the molecular mechanisms underlying constitutive activation of Stat5a/b in prostate cancer are unclear. The receptor-associated tyrosine kinase Jak2 is a known key activator of Stat5a/b in prostate cancer cells in response to ligand stimulation. Recently, a single gain-of-function point mutation of JAK2 was described in myeloproliferative diseases leading to constitutive Jak2 kinase activity, subsequent Stat5a/b activation and involvement of V617F Jak2 in the pathogenesis of myeloproliferative disorders.Materials and Methods: We determined whether JAK2 undergoes the V617F activating mutation during clinical progression of human prostate cancer using a highly sensitive assay (amplification refractory mutation system) and a unique material of fresh specimens from organ-confined or castration-resistant prostate cancers.Results: The JAK2 V617F mutation was not found in any of the normal or malignant prostate samples analyzed in this study.Conclusions: Future work should focus on determining the molecular mechanisms other than V617F mutation of Jak2 resulting in continuous Stat5 activation in clinical prostate cancers.

Expression of the actin-associated protein transgelin (SM22) is decreased in prostate cancer

Transgelin is an actin-binding protein shown to be tumour-suppressive. Loss of transgelin expression in transformed cells is associated with oncogenesis. This study aimed to determine whether transgelin expression was suppressed in prostate cancer. An in silico meta-analysis with public-domain expressed-sequence-tag libraries of normal human prostate epithelium, prostatic intraepithelial neoplasia, invasive carcinoma and metastasised lesions predicted decreased transgelin expression with disease progression. Similarly, analysis of Affymetrix gene chip data and the Oncomine database indicated that transgelin was one the 2% most significant of all down-regulated genes in response to prostate cancer. Analysis by quantitative reverse transcription with the polymerase chain reaction (qRT-PCR) of patient biopsies determined transgelin expression to be significantly lower in prostate tumour tissue than in matched normal tissue. Similarly, qRT-PCR and Western blot analysis of representative prostate cancer cell lines demonstrated significantly lower levels of transgelin mRNA and protein in all but the DU145 prostate cancer cell line. Increased expression of TAGLN and increased transgelin protein in response to treatment with transforming growth factor-beta suggested that reduced expression in prostate cancer was not attributable to gene promoter suppression by hypermethylation. Gene ontology function analysis highlighted the importance of transgelin in the co-deregulation of actin-binding proteins. Thus, transgelin is suppressed during prostate cancer progression and seems to be an important factor in the dysregulation of the actin cytoskeleton.