Differentiation Of Prostate Epithelial Cells Research Articles

Abstract B006: Identifying the molecular signature of bi-potent prostate epithelial cells through scRNA-seq

Abstract Introduction: Recent studies have shown that cells undergoing oncogenic processes usurp normal developmental pathways to escape regulators of unsolicited growth. Basal to luminal differentiation in the prostate serves as another cellular process that can go awry, the dysregulation of which can enable oncogenesis in the prostate. Studies have found bi-potent cells in prostate epithelium and speculate those to be cells of origin in prostate cancer. Methods and Results: Through single-cell RNA sequencing we investigated basal to luminal differentiation of normal human prostate epithelial cells across timepoints Days 0, 4, 8, 12, 16, and 20 by stimulating with FGF-7 (20ng/mL) and R1881 (10nM) and explored the data to identify unique populations present in the differentiation process. Our results show spatial separation at various timepoints with distinct transcriptomics profile at Day 0, Day 16, and Day 20. As expected, populations present at Day 0 are also present across all timepoint, though enrichment of various distinct populations emerged throughout the differentiation process. Using trajectory analysis (Monocle3) and the Cell Surface Protein Atlas, we profiled surface proteins for each cluster at Day 0 and identified a set of unique surface markers (SLC3A, LYPD3, TACSTD2, DSC2) from undifferentiated basal cells that express both basal (TP63+, K14+) and luminal markers (K8+, K18+), potentially serving as a bi-potent prostate epithelial cells. Conclusion: Our data have identified unique populations present in our prostate differentiation model expressing both luminal and basal signatures which can be isolated using identified surface markers to investigate the role of bi-potent cells in prostate differentiation and oncogenesis. Citation Format: Hunain Khawaja, Cynthia Miranti. Identifying the molecular signature of bi-potent prostate epithelial cells through scRNA-seq [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B006.

Abstract 4720: Chromatin conformation alterations in prostate cancer progression and formation of castration resistance

Abstract Introduction: Prostate cancer (PC) is the most common cancer in men and second in cancer related deaths. Aggressive prostate cancer is typically treated with androgen depletion therapy which often results in the formation of castration resistant prostate cancer (CRPC). Androgen receptor (AR) is a transcription factor that regulates hundreds of target genes and is responsible for the differentiation and function of normal prostate epithelial cells. In addition to AR, multiple other TFs that bind to distal enhancers have been implicated in PC, such as FOXA1, MYC and HOXB13. Understanding the role of these distal regulators is paramount in PC. Methods: To study the changes in chromatin conformation in prostate cancer progression, we have sequenced clinical benign prostatic hyperplasia (BPH), PC and CRPC samples using Hi-C sequencing. From each group 6 samples were sequenced with a total of 18 samples in the cohort. In addition, we have combined a multi omics cohort consisting of chromatin accessibility, transcriptome and methylome sequencing data from matching samples. Together these data types allow us to map out the regulatory networks in PC and CRPC by probing the distal active regulatory sites and their interactions. To account for artifacts caused by genomic aberrations in cancer samples we have established a pipeline for normalizing these effects in HiC-data. Results: Overall topologically associated domains (TADs) and A/B-compartments remain stable between the groups. However, local changes of compartments in loci of key genes, such as AKR1C3 and FOXA1 correlate with their gene expression. We detect differential loops between sample groups and these changes are associated with gene expression alterations in PCs. Distal enhancers of PC up-regulated genes are enriched for FOXA1 and HOXB13. Interestingly, up-regulated loops in CRPC are enriched for FOXA1 and HOXB13, while no systematic effect on gene expression is seen on a genome wide scale, apart from genes such as AR and CENPE whose expression correlates strongly with their association to distal enhancers. We also detect enhancer hijacking events in driver genes. One example is the recurrent ETV1-MIPOL1 fusion, where promoter of ETV1 forms contacts with FOXA1 enhancer regions. Conclusions: Alterations in loop structures are strongly associated with regulatory programs that have been previously implicated in PC, including regulation by FOXA1, HOXB13 and other transcription factors. This highlights the importance of chromatin conformation in facilitating the effects of regulatory programs that rely on distal enhancers in PC. These changes appear more systematic in untreated PCs, whilst CRPCs exhibit more heterogeneity in chromatin conformation across the samples. Citation Format: Tuomo Virtanen, Ebrahim Afyounian, Riikka Nurminen, Teuvo Tammela, Tapio Visakorpi, Juha Kesseli, Kirsi Rautajoki, Matti Nykter. Chromatin conformation alterations in prostate cancer progression and formation of castration resistance. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4720.

Abstract 869: Post-transcriptional control of androgen receptor expression

Abstract The Androgen Receptor (AR) is a ligand-dependent transcription factor that modulates the expression of androgen-regulated genes involved in the proliferation and differentiation of normal prostate epithelial cells. However, in Prostate Cancer (PCa), these cellular events are dysregulated upon significant changes in AR, such as AR overexpression, leading to uncontrolled cell growth and proliferation. To elucidate the molecular mechanisms by which AR expression and function are controlled, we have explored the post-transcriptional regulatory RNA elements of AR, particularly the 3’ untranslated region (UTR). The AR 3’UTR is 6.8 Kilobases (Kb) long, which indicates that this region is likely a site for multitudinous levels of post-transcriptional regulation. We found that the length of AR 3’UTR negatively correlates with AR expression, with the full length 3’UTR leading to the most downregulation of expression. Currently, we are exploring cis-acting noncoding RNAs and RNA binding proteins (RBPs) predicted to interact with the AR 3’UTR. Citation Format: Eviania Likos, Girish C. Shukla. Post-transcriptional control of androgen receptor expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 869.

Relationship between endogenous testosterone and prostate carcinoma.

Prostate cancer incidence is rising rapidly worldwide, which includes Asian countries too. There are a lot of controversies regarding the link between endogenous testosterone levels and prostate disorders. This study was conducted to understand the value of serum testosterone in predicting the risk and occurrence of prostate carcinoma. Eighty males in the age group of 50-80 years, out of which forty were diagnosed as Prostate cancer patients and forty were healthy controls matched with respect to age and sex, were recruited in the study. Serum testosterone and serum prostate-specific antigen levels were estimated in both groups. Serum testosterone levels were low in 67.5% and normal in 32.5% of the patients of prostate cancer. In comparison with the controls, serum PSA levels were observed to be higher in prostate cancer patients (P < 0.001). Analysis of the data was done with the help of an unpaired t-test, and P < 0.05 was considered to be statistically significant. Androgens promote cellular differentiation and proliferation of prostate epithelial cells via the genetic alterations in the TMPRSS2 gene and ETS (E26 transforming sequences) transcription factor genes involved in cell proliferation and tumor cell invasion. These genes are found to be over-expressed in prostate carcinomas. Suppression of testosterone by prostate cancer-induced production of inhibin that initiates the hypothalamic-pituitary axis negative feedback mechanism might be the reason for lower levels of serum testosterone in prostate carcinoma patients. Hence, screening of elderly men above 60 years of age for serum testosterone levels should be done for early identification of prostate carcinoma as well as a better prognosis in the management of the disease.

USE OF TESTOSTERONE REPLACEMENT THERAPY AFTER RADICAL PROSTATECTOMY MIGHT KILL TWO BIRDS WITH ONE STONE FROM THE PERSPECTIVE OF MEN’S HEALTH AND DISEASE CONTROL

Testosterone plays an important role in promoting the differentiation and stimulation of prostate epi-thelial cells. Adding to the basic physiology of testosterone, several studies led to the dissemination of the historical androgen hypothesis that higher circulating androgen levels promote prostate cancer cell growth and make the tumor more aggressive. Whereas, the prostate saturation model accounted for the androgen sensitivity of testosterone stimulation in the prostate up to a saturation point, which occurred near the level of castration. Testosterone is related to prostate cancer metabolism in a com-plex manner; however, within the reference range, high or normal testosterone levels are expected to maintain benign and malignant prostate cells in a differentiated state. Accumulating evidence has suggested that testosterone replacement therapy (TRT) might prevent the development of prostate cancer and even reduce prostate cancer risk. There is no change in the clinical guidelines to be followed when considering TRT in patients with a history of prostate cancer, but it is necessary for the physician to inform patients of the positive effects of TRT on male health and prostate cancer.

PARP Inhibition Suppresses GR-MYCN-CDK5-RB1-E2F1 Signaling and Neuroendocrine Differentiation in Castration-Resistant Prostate Cancer.

In this study, we addressed the underlying mechanisms for the association between enzalutamide (ENZ) treatment and neuroendocrine prostate cancer (NEPC), and the critical involvement of MYCN, and loss of RB1 function in neuroendocrine differentiation (NED) of prostatic epithelial cells, and the development of NEPC. We further sought to determine whether PARP inhibition could suppress NEPC, and to identify molecular determinants of this therapeutic activity. We used a novel prostate cancer patient-derived xenograft (PDX) treatment model, prostatic adenocarcinoma and NEPC cell lines, an NEPC organoid line, and NEPC xenograft models to address the mechanistic basis of ENZ-induced NED, and to analyze suppression of NED and NEPC growth by PARP inhibition. We identified an ENZ treatment-associated glucocorticoid receptor (GR)-MYCN-CDK5-RB1-E2F1 signaling pathway that drives NED in prostatic adenocarcinoma PDX and cell line models. Mechanistically, long-term ENZ treatment transcriptionally upregulates signaling of the GR-MYCN axis, leading to CDK5R1 and CDK5R2 upregulation, Rb1 phosphorylation, and N-Myc-mediated and E2F1-mediated NED gene expression. Importantly, olaparib (OLA) or talazoparib (TALA) suppressed these activities, and the combination of OLA and dinaciclib (DINA), an inhibitor of CDK2 and CDK5, which also inhibits Rb1 phosphorylation, suppressed NED and significantly improved therapeutic efficiency in NEPC cells in vitro and in NEPC tumors in vivo. The results of our study indicate an important role of GR-MYCN-CDK5R1/2-RB1-NED signaling in ENZ-induced and PARP inhibitor-suppressed NEPC. We also demonstrated efficacy for OLA+DINA combination therapy in NEPC xenograft models.

Notch signalling is a potential resistance mechanism of progenitor cells within patient-derived prostate cultures following ROS-inducing treatments.

Low Temperature Plasma (LTP) generates reactive oxygen and nitrogen species, causing cell death, similarly to radiation. Radiation resistance results in tumour recurrence, however mechanisms of LTP resistance are unknown. LTP was applied to patient‐derived prostate epithelial cells and gene expression assessed. A typical global oxidative response (AP‐1 and Nrf2 signalling) was induced, whereas Notch signalling was activated exclusively in progenitor cells. Notch inhibition induced expression of prostatic acid phosphatase (PAP), a marker of prostate epithelial cell differentiation, whilst reducing colony forming ability and preventing tumour formation. Therefore, if LTP is to be progressed as a novel treatment for prostate cancer, combination treatments should be considered in the context of cellular heterogeneity and existence of cell type‐specific resistance mechanisms.

Abstract 1900: Understanding the biological role of latexin in the normal and malignant prostate

Abstract The retinoid-inducible protein Latexin (LXN) acts as a stem cell suppressor in normal murine hematopoiesis, and in human tumors where it can influence both tumor cell migration and invasion (Liang et al, Nature genetics 2007). We have shown that LXN is silenced in the stem cell fraction of primary prostate epithelial cells, and that expression increases as cells differentiate through the prostate basal epithelial hierarchy towards mature luminal cells. Furthermore, transient knockdown of LXN significantly increased clonogenic potential and promoted a more invasive phenotype in primary prostate epithelia (Oldridge et al, Oncogenesis 2013). LXN may therefore be involved in the differentiation of prostate epithelial cells, but could act as a tumor suppressor in Prostate Cancer (CaP), in addition to other tumours. In genome databases LXN is described as an inhibitor for Carboxypeptidase A4, but despite considerable molecular characterisation, the mechanism of action has remained an enigma since perhaps paradoxically LXN appears to be able to function independently of its classical CPA4 inhibitory role. To resolve this, we adopted an unbiased approach to determine if targeted re-expression of LXN or its binding partners could provide a viable novel drug target for the treatment of CaP. Our preliminary studies had revealed that LXN was an exclusively cytosolic protein in prostate epithelial cells. However, subsequent live cell imaging of primary prostate epithelial cells overexpressing YFP-LXN demonstrated for the first time that, whilst predominantly cytosolic, LXN was also present within the cell nucleus. To characterise the effects of LXN overexpression on gene transcription after LXN-expressing lentiviral transduction of primary prostate epithelial cells, we utilised Affymetrix HTA2 gene arrays. Perhaps surprisingly, short-term overexpression of LXN did not significantly effect global gene transcription compared to controls (n=4 P=NS). Additionally, we used IP/MS to investigate the potential interacting partners of LXN, using multiple approaches, including both overexpression of HA-tagged and WT LXN, and endogenous LXN as bait. These approaches failed to reveal any strong (and reproducible) intracellular protein interactors of LXN. From these data, we hypothesised that LXN may function as a secreted protein. We found that LXN was present in not only the conditioned media of LXN+ prostate epithelial cells, but also preliminary evidence that LXN is enriched in extracellular exosomes. Therefore, LXN may perform its biological roles extracellularly, or at the cell surface. Our current work is focusing on determining the functional consequences of secreted LXN on prostate epithelia and to establish potential protein: protein interactions of LXN in the extracellular space. Full characterisation of LXN function considering its potent biological effects on cancer cells will determine its suitability as a therapeutic in CaP and other cancers. Citation Format: Robert I. Seed, Alberto J. Taurozzi, Giovanna Nappo, Holger H. Erb, Anne Collins, Norman J. Maitland, Fiona Frame. Understanding the biological role of latexin in the normal and malignant prostate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1900. doi:10.1158/1538-7445.AM2017-1900

Downregulation of CPA4 to suppress NSCLC proliferation by inducing apoptosis and G1 arrest.

e20074 Background: Carboxypepidase A4 (CPA4) is a member of the metallocarboxypeptidase family. Previous study discovered that CPA4 may participate in cell growth and differentiation of prostate epithelial cells. Meanwhile, CPA4 is a printed gene and thought to be involved in prostate cancer aggressiveness. As is reported, CPA4 was increased in NSCLC tissues compared to normal lung tissues and high expression of CPA4 was correlated with poor prognosis of NSCLC patients. However, the role of CPA4 play in lung tumorigenesis is still unclear. Methods: We examined the mRNA and protein expression level of CPA4 via real-time PCR and immunohistochemistry in NSCLC tissues and adjacent tissues. Growth assays both in vitro and in vivo were performed to elucidate the role of CPA4 may play in lung cancer and Fluorescence Activated Cell Sorter was conducted to uncover the putative mechanism. Results: CPA4 expression was increased both in mRNA and protein levels in NSCLC tissues compared to adjacent tissues. MTT and colony formation assays showed that downregulation of CPA4 in H1299 and A549 cells inhibited lung cancer cells proliferation. We further confirmed this result by using cellomics and celligo. Depleting CPA4 also suppressed tumor growth in mice. Mechanically, we found that suppressing CPA4 expression in lung cancer cells could induce apoptosis and G1 arrest. We supposed that CPA4 expression may be associated with caspase family and it needs further studies. Conclusions: Collectively, we demonstrate that decreased CPA4 inhibits NSCLC proliferation via inducing apoptosis and G1 arrest.

Crosstalk between the Androgen Receptor and PPAR Gamma Signaling Pathways in the Prostate.

Nuclear receptors are a superfamily of ligand-activated transcription factors that play critical roles in the regulation of normal biological processes and several disease states. Of the nuclear receptors expressed within the prostate, the androgen receptor (AR) promotes the differentiation of prostatic epithelial cells and stimulates production of enzymes needed for liquefaction of semen. Multiple forms of AR also promote the growth of both early and late stage prostate cancers. As a result, drugs that target the AR signaling pathway are routinely used to treat patients with advanced forms of prostate cancer. Data also suggest that a second member of the nuclear receptor superfamily, the peroxisome proliferator activated receptor gamma (PPARγ), is a tumor suppressor that regulates growth of normal prostate and prostate cancers. Recent studies indicate there is a bidirectional interaction between AR and PPARγ, with each receptor influencing the expression and/or activity of the other within prostatic tissues. In this review, we examine how AR and PPARγ each regulate the growth and development of normal prostatic epithelial cells and prostate cancers. We also discuss interactions between the AR and PPARγ signaling pathways and how those interactions may influence prostate biology.

RET-mediated glial cell line-derived neurotrophic factor signaling inhibits mouse prostate development.

In humans and rodents, the prostate gland develops from the embryonic urogenital sinus (UGS). The androgen receptor (AR) is thought to control the expression of morphogenetic genes in inductive UGS mesenchyme, which promotes proliferation and cytodifferentiation of the prostatic epithelium. However, the nature of the AR-regulated morphogenetic genes and the mechanisms whereby AR controls prostate development are not understood. Glial cell line-derived neurotrophic factor (GDNF) binds GDNF family receptor α1 (GFRα1) and signals through activation of RET tyrosine kinase. Gene disruption studies in mice have revealed essential roles for GDNF signaling in development; however, its role in prostate development is unexplored. Here, we establish novel roles of GDNF signaling in mouse prostate development. Using an organ culture system for prostate development and Ret mutant mice, we demonstrate that RET-mediated GDNF signaling in UGS increases proliferation of mesenchyme cells and suppresses androgen-induced proliferation and differentiation of prostate epithelial cells, inhibiting prostate development. We also identify Ar as a GDNF-repressed gene and Gdnf and Gfrα1 as androgen-repressed genes in UGS, thus establishing reciprocal regulatory crosstalk between AR and GDNF signaling in prostate development.

The ETS factor ESE3/EHF represses IL-6 preventing STAT3 activation and expansion of the prostate cancer stem-like compartment

Metastatic prostate cancer represents a yet unsolved clinical problem due to the high frequency of relapse and treatment resistance. Understanding the pathways that lead to prostate cancer progression is an important task to prevent this deadly disease. The ETS transcription factor ESE3/EHF has an important role in differentiation of human prostate epithelial cells. Loss of ESE3/EHF in prostate epithelial cells determines transformation, epithelial-to-mesenchymal transition (EMT) and acquisition of stem-like properties. In this study we identify IL-6 as a direct target of ESE3/EHF that is activated in prostate epithelial cells upon loss of ESE3/EHF. ESE3/EHF and IL-6 were significantly inversely correlated in prostate tumors. Chromatin immunoprecipitation confirmed binding of ESE3/EHF to a novel ETS binding site in the IL-6 gene promoter. Inhibition of IL-6 reverted transformation and stem-like phenotype in tumorigenic ESE3/EHF knockdown prostate epithelial cell models. Conversely, IL-6 stimulation induced malignant phenotypes, stem-like behavior and STAT3 activation. Increased level of IL-6 was observed in prostatospheres compared with adherent bulk cancer cells and this was associated with stronger activation of STAT3. Human prostate tumors with IL-6 elevation and loss of ESE3/EHF were associated with STAT3 activation and displayed upregulation of genes related to cell adhesion, cancer stem-like and metastatic spread. Pharmacological inhibition of IL-6/STAT3 activation by a JAK inhibitor restrained cancer stem cell growth in vitro and inhibited self-renewal in vivo. This study identifies a novel connection between the transcription factor ESE3/EHF and the IL-6/JAK/STAT3 pathway and suggests that targeting this axis might be preferentially beneficial in tumors with loss of ESE3/EHF.

Androgen receptor differentially regulates the proliferation of prostatic epithelial cells in vitro and in vivo.

Androgens regulate the proliferation and differentiation of prostatic epithelial cells, including prostate cancer (PCa) cells in a context-dependent manner. Androgens and androgen receptor (AR) do not invariably promote cell proliferation; in the normal adult, endogenous stromal and epithelial AR activation maintains differentiation and inhibits organ growth. In the current study, we report that activation of AR differentially regulates the proliferation of human prostate epithelial progenitor cells, NHPrE1, in vitro and in vivo. Inducing AR signaling in NHPrE1 cells suppressed cell proliferation in vitro, concomitant with a reduction in MYC expression. However, ectopic expression of AR in vivo stimulated cell proliferation and induced development of invasive PCa in tissue recombinants consisting of NHPrE1/AR cells and rat urogenital mesenchymal (UGM) cells, engrafted under renal capsule of adult male athymic mice. Expression of MYC increased in the NHPrE1/AR recombinant tissues, in contrast to the reduction seen in vitro. The inhibitory effect of AR signaling on cell proliferation in vitro were reduced by co-culturing NHPrE1/AR epithelial cells with prostatic stromal cells. In conclusion, these studies revealed that AR signaling differentially regulates proliferation of human prostatic epithelia cells in vitro and in vivo through mechanisms involving stromal/epithelial interactions.

Abstract 1824: Context dependent regulatory patterns of the androgen receptor (AR) and androgen receptor target genes

Abstract Background: Expression of the androgen receptor (AR) is associated with androgen-dependent proliferation arrest and terminal differentiation of normal prostate epithelial cells. Additionally, activation of the AR is required for survival of benign luminal epithelial cells and primary cancer cells. Androgen deprivation therapy (ADT) leads to apoptosis in both benign and cancerous tissue. Cancer cell escape from ADT is known as castration-resistant prostate cancer (CRPC). In the course of CRPC development the AR typically switches from being a cell-intrinsic inhibitor of normal prostate epithelial cell proliferation to becoming an oncogene that is critical for prostate cancer cell proliferation. Methods: Immortalized human prostate basal epithelial EP156T cells and progeny cells that underwent epithelial to mesenchymal transition (EMT), primary prostate epithelial cells (PrECs) and prostate cancer cell lines LNCaP, VCaP and 22Rv1 were used to examine context dependent restriction and activation of the AR and classical target genes, such as KLK3. Genome-wide gene expression analyses and single cell protein analyses were applied to study the effect of different contexts. Results: A variety of growth conditions were tested and found unable to activate AR expression and transcription of classical androgen-dependent AR target genes, such as KLK3, in prostate epithelial cells with basal cell features or in mesenchymal type prostate cells. The restriction of androgen and AR dependent transcription of classical target genes in prostate basal epithelial cells was at the level of AR expression. Exogenous AR expression was sufficient for androgen-dependent transcription of AR target genes in prostate basal epithelial EP156T-AR cells, but did not exert a positive feedback on endogenous AR expression. Mesenchymal type prostate EPT3-AR cells with exogenous AR expression, in contrast to epithelial type EP156T-AR cells, were androgen non-responsive and were unable to produce detectable PSA in the culture supernatants even with higher levels of exogenous AR protein than in EP156T-AR and LNCaP cells for up to 2 weeks in androgen containing growth medium. The restricted PSA expression in the mesenchymal context suggests that if ADT increases the pool of mesenchymal type prostate cancer cells, then this might go undetected during PSA monitoring of disease progression. Citation Format: Jan Roger Olsen, Waqas Azeem, Margrete R. Hellem, Kristo Marvyin, Yaping Hua, Yi Qu, Lisha Li, Biaoyang Lin, XiSong Ke, Anne Margrete Oyan, Karl-Henning Kalland. Context dependent regulatory patterns of the androgen receptor (AR) and androgen receptor target genes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1824.

Context dependent regulatory patterns of the androgen receptor and androgen receptor target genes.

BackgroundExpression of the androgen receptor (AR) is associated with androgen-dependent proliferation arrest and terminal differentiation of normal prostate epithelial cells. Additionally, activation of the AR is required for survival of benign luminal epithelial cells and primary cancer cells, thus androgen deprivation therapy (ADT) leads to apoptosis in both benign and cancerous tissue. Escape from ADT is known as castration-resistant prostate cancer (CRPC). In the course of CRPC development the AR typically switches from being a cell-intrinsic inhibitor of normal prostate epithelial cell proliferation to becoming an oncogene that is critical for prostate cancer cell proliferation. A clearer understanding of the context dependent activation of the AR and its target genes is therefore desirable.MethodsImmortalized human prostate basal epithelial EP156T cells and progeny cells that underwent epithelial to mesenchymal transition (EMT), primary prostate epithelial cells (PrECs) and prostate cancer cell lines LNCaP, VCaP and 22Rv1 were used to examine context dependent restriction and activation of the AR and classical target genes, such as KLK3. Genome-wide gene expression analyses and single cell protein analyses were applied to study the effect of different contexts.ResultsA variety of growth conditions were tested and found unable to activate AR expression and transcription of classical androgen-dependent AR target genes, such as KLK3, in prostate epithelial cells with basal cell features or in mesenchymal type prostate cells. The restriction of androgen- and AR-dependent transcription of classical target genes in prostate basal epithelial cells was at the level of AR expression. Exogenous AR expression was sufficient for androgen-dependent transcription of AR target genes in prostate basal epithelial cells, but did not exert a positive feedback on endogenous AR expression. Treatment of basal prostate epithelial cells with inhibitors of epigenetic gene silencing was not efficient in inducing androgen-dependent transcription of AR target genes, suggesting the importance of missing cofactor(s).ConclusionsRegulatory mechanisms of AR and androgen-dependent AR target gene transcription are insufficiently understood and may be critical for prostate cancer initiation, progression and escape from standard therapy. The present model is useful for the study of context dependent activation of the AR and its transcriptome.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2453-4) contains supplementary material, which is available to authorized users.

Regulation of Prostate Development and Benign Prostatic Hyperplasia by Autocrine Cholinergic Signaling via Maintaining the Epithelial Progenitor Cells in Proliferating Status.

SummaryRegulation of prostate epithelial progenitor cells is important in prostate development and prostate diseases. Our previous study demonstrated a function of autocrine cholinergic signaling (ACS) in promoting prostate cancer growth and castration resistance. However, whether or not such ACS also plays a role in prostate development is unknown. Here, we report that ACS promoted the proliferation and inhibited the differentiation of prostate epithelial progenitor cells in organotypic cultures. These results were confirmed by ex vivo lineage tracing assays and in vivo renal capsule recombination assays. Moreover, we found that M3 cholinergic receptor (CHRM3) was upregulated in a large subset of benign prostatic hyperplasia (BPH) tissues compared with normal tissues. Activation of CHRM3 also promoted the proliferation of BPH cells. Together, our findings identify a role of ACS in maintaining prostate epithelial progenitor cells in the proliferating state, and blockade of ACS may have clinical implications for the management of BPH.

Abstract B23: Epigenetic control of prostate epithelial stem cell differentiation

Abstract Introduction: Hormone manipulation offers an effective but short term and time-limited treatment for human prostate cancers, reducing both tumor bulk and serum levels of prostate-specific antigen. In normal prostate, castration results in a loss of luminal cells and persistence of a poorly differentiated basal-like population, which is responsible for repopulation after restoration of hormone levels. However in fetal prostate development, the hormonal stimulus is provided indirectly to the developing epithelial structures via androgen-sensitive stromal cells. To study the drivers of prostate epithelial cell differentiation, we sought the epigenetic factors affecting cell fate in adult human normal, hyperplastic and malignant epithelium, to provide more precise targets for prostate cancer therapy, based on this developmental paradigm. Methods: We have adopted a multiple model approach for the study of human prostate stem cells. Epithelial cell fractions are prepared from human tissues using multiparametric FACS with a Beckman Coulter Astrios, or adhesion-based separations from (i) MRI guided biopsies of prostate tissues (and patient matched non- tumor tissues) (ii) primary cultures in defined serum free medium from prostate tissues depleted of stromal cells and (iii) a novel set of near patient mouse xenografts of human tissues. In these systems the (normal) colony forming and (malignant) tumor-inducing capacity largely resides in a CD133+/ CD44+/a2B1 integrin Hi/CD49f population which comprises 1% and &amp;lt;0.1% of the normal and malignant epithelium respectively. Cell populations were analyzed independently for their gene expression patterns, non-coding RNA expression, histone modifications and genome CpG methylation. Using a modified analysis algorithm we then related gene expression to both miRNA expression and genomic methylation/ chromatin condensation. Results: At the chromosomal level, prostate SC display a unique pattern of CpG methylation compared to their more differentiated progeny, with distinct changes in cancer. The SC also have a characteristic bivalent chromatin pattern when probed for specific histone modifications. This bivalent chromatin pattern is also indicative, in embryonic systems, of monoallelic gene expression, which was confirmed in prostate SC populations by analysis of the prostate-specific TMPRSS2 gene, in both native and fused form (to ERG). MicroRNA expression patterns are tightly linked to SC differentiation, rather than carcinogenesis, and the miRNAs expressed in prostate SC most closely resemble those in human embryonic SC, and intriguingly in the most malignant forms of prostate cancer. Since miRNAs can control expression of multiple genes, we have devised an algorithm to predict regulation of only those genes expressed in the prostate epithelium. Perhaps significantly for cancer treatment, the principal functions controlled by miRNA expression are both DNA damage repair and wnt signaling, manipulation of which can increase the radiation resistance resident in the SC population. By studying genes which are tightly co-regulated during cell differentiation, we defined 4 non-overlapping sets of genes in which retinoic acid acts as a driver of primitive cell differentiation, an intermediate cell type expressing genes with a bivalent response to RA and androgens, and committed basal/luminal cells in which androgen control of cell fate dominates. Discussion: By studying highly enriched cell populations in different model systems, all of which are derived from primary human tissues, we have built up a complex picture of epigenetic regulation of gene expression during differentiation of human prostate. Many of these essential functions are related to similar functions in developing prostate tissue, but are often characterized by phenotypic plasticity, where the differentiated products of asymmetric division (self-renewal) can revert to a stem-like state. Citation Format: Norman James Maitland, John Packer. Epigenetic control of prostate epithelial stem cell differentiation. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B23.

MP04-14 PHASE IIA, RANDOMIZED PLACEBO-CONTROLLED TRIAL OF SINGLE HIGH DOSE CHOLECALCIFEROL (VITAMIN D3) AND DAILY GENISTEIN (G-2535) VERSUS PLACEBO IN MEN WITH EARLY STAGE PROSTATE CANCER UNDERGOING PROSTATECTOMY

You have accessJournal of UrologyProstate Cancer: Epidemiology & Natural History I1 Apr 2016MP04-14 PHASE IIA, RANDOMIZED PLACEBO-CONTROLLED TRIAL OF SINGLE HIGH DOSE CHOLECALCIFEROL (VITAMIN D3) AND DAILY GENISTEIN (G-2535) VERSUS PLACEBO IN MEN WITH EARLY STAGE PROSTATE CANCER UNDERGOING PROSTATECTOMY David F. Jarrard, Badrinath R. Konety, Joel Slaton, Wei Huang, Tracy Downs, Jill Kolesar, KyungMann Kim, Tom Havighurst, Margaret G. House, Howard L. Parnes, and Howard H. Bailey David F. JarrardDavid F. Jarrard More articles by this author , Badrinath R. KonetyBadrinath R. Konety More articles by this author , Joel SlatonJoel Slaton More articles by this author , Wei HuangWei Huang More articles by this author , Tracy DownsTracy Downs More articles by this author , Jill KolesarJill Kolesar More articles by this author , KyungMann KimKyungMann Kim More articles by this author , Tom HavighurstTom Havighurst More articles by this author , Margaret G. HouseMargaret G. House More articles by this author , Howard L. ParnesHoward L. Parnes More articles by this author , and Howard H. BaileyHoward H. Bailey More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1949AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Prostate cancer (PCa) represents an important target for chemoprevention given its prolonged natural history and predilection for afflicting an older population. Epidemiologic and laboratory data suggest that vitamin D and genistein (soy) in decreasing PCa progression. The effect of vitamin D on prostate epithelial cell proliferation and differentiation is well documented and genistein may augment this affect through inhibition of the CYP24 enzyme, which is responsible for intracellular vitamin D metabolism. In addition, both genistein and vitamin D inhibit the intraprostatic synthesis of prostaglandin E2, an important mediator of inflammation. In the current prospective, multicenter trial in patients undergoing radical prostatectomy for localized prostate cancer, the safety and effect of adding genistein to patients receiving a single, large dose of cholecalciferol was assessed. METHODS Men undergoing radical prostatectomy were randomly assigned to one of two treatment groups: (1) cholecalciferol: 200,000 IU as one dose at study entry plus genistein: 600 mg daily or (2) placebo cholecalciferol day 1 and placebo genistein PO daily for 21-28 days prior to radical prostatectomy. Serum and tissue analyses were performed and side-effects recorded. RESULTS A total of 15 patients were enrolled, 8 in the placebo arm and 7 in the vitamin D + genistein (VD+G) arm. All patients were compliant and completed the study. No significant differences in side effect profiles were noted. Utilization of the VD+G showed a trend toward increased calcitriol serum concentrations when compared to placebo (0.104 +/- 0.1 vs. 0.0013 +/-0.07; p=0.08); however, prostate tissue levels did not increase. Immunohistochemistry for marker analyses using VECTRA automated quantitation revealed a significant increase in AR expression (p=0.04) and a trend toward increased TUNEL staining (p=0.1) in prostate cancer tissues in men randomized to receive VD+G compared to placebo. CONCLUSIONS In this first study testing the combination of a single, large dose of cholecalciferol (200,000IU) and daily genistein (G-2535) the agents were well tolerated. While serum levels of calcitriol were increased on the intervention arm, prostate tissue levels were not. Increased prostatic AR expression (suggesting increased differentiation) and a trend toward increased TUNEL staining (increased apoptosis) were observed among men randomized to the combination. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e33-e34 Advertisement Copyright & Permissions© 2016MetricsAuthor Information David F. Jarrard More articles by this author Badrinath R. Konety More articles by this author Joel Slaton More articles by this author Wei Huang More articles by this author Tracy Downs More articles by this author Jill Kolesar More articles by this author KyungMann Kim More articles by this author Tom Havighurst More articles by this author Margaret G. House More articles by this author Howard L. Parnes More articles by this author Howard H. Bailey More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Multi-Drug Resistance ABC Transporter Inhibition Enhances Murine Ventral Prostate Stem/Progenitor Cell Differentiation.

Multi-drug resistance (MDR)-ATP binding cassette (ABC) transporters, ABCB1, ABCC1, and ABCG2 participate in the efflux of steroid hormones, estrogens, and androgens, which regulate prostate development and differentiation. The role of MDR-ABC efflux transporters in prostate epithelial proliferation and differentiation remains unclear. We hypothesized that MDR-ABC transporters regulate prostate differentiation and epithelium regeneration. Prostate epithelial differentiation was studied using histology, sphere formation assay, and prostate regeneration induced by cycles of repeated androgen withdrawal and replacement. Embryonic deletion of Abcg2 resulted in a decreased number of luminal cells in the prostate and increased sphere formation efficiency, indicating an imbalance in the prostate epithelial differentiation pattern. Decreased luminal cell number in the Abcg2 null prostate implies reduced differentiation. Enhanced sphere formation efficiency in Abcg2 null prostate cells implies activation of the stem/progenitor cells. Prostate regeneration was associated with profound activation of the stem/progenitor cells, indicating the role of Abcg2 in maintaining stem/progenitor cell pool. Since embryonic deletion of Abcg2 may result in compensation by other ABC transporters, pharmacological inhibition of MDR-ABC efflux was performed. Pharmacological inhibition of MDR-ABC efflux enhanced prostate epithelial differentiation in sphere culture and during prostate regeneration. In conclusion, Abcg2 deletion leads to activation of the stem/progenitor cells and enhances differentiating divisions; and pharmacological inhibition of MDR-ABC efflux leads to epithelial differentiation. Our study demonstrates for the first time that MDR-ABC efflux transporter inhibition results in enhanced prostate epithelial cell differentiation.

Abstract 1451: MicroRNAs regulated by ESE3/EHF control important mediators of epithelial cell differentiation and stemness in prostate tumors

Abstract Deregulated expression of ETS transcription factors has emerged as an important event in prostate cancer pathogenesis. We found that loss of the ETS factor ESE3/EHF induced a broad dedifferentiation program associated with epithelial-to-mesenchymal transition (EMT) and induction of metastatic and cancer stem-like cell properties. To understand the mechanism by which ESE3/EHF controls differentiation of prostate epithelial cells, we examined microRNA (miRNA) expression in a cohort of primary prostate tumors and prostate epithelial normal and cancer cells using miRNA gene arrays. A distinct set of miRNAs was specifically deregulated in cancer cells and tumors with low ESE3/EHF expression. Bioinformatic analysis indicated that the deregulated miRNAs controlled many genes involved in EMT and cell stemness. Interestingly, we found that miR-424 was at the top list of the miRNAs up-regulated in ESE3 low expressing tumors and cell lines. This finding was confirmed by qRT-PCR both in cells and human tumors. Functional assays showed that ESE3/EHF controlled directly miR-424 by binding to the pre-miRNA promoter and repressing its transcription. Inhibition of miR-424 using an antimiR reduced anchorage-independent growth and cell migration in cancer cells with low ESE3 expression and high miR-424 level. On the contrary, stable expression of pre-miR-424 in cells with low endogenous miR-424 level increased anchorage-independent growth and cell migration. Furthermore, modulation of miR-424 expression affected in vitro prostatosphere formation, a phenotype associated with cancer stem-like cell properties. Consistently, inhibition of miR-424 in DU145 prostate cancer cells reduced growth of tumor xenografts in immunodeficient mice. Integrating bioinformatic analyses of the predicted targets and gene profiling of cells with miR-424 overexpression we found that miRNA-424 controlled several factors involved in protein degradation. Collectively, these results show for the first time that ESE3/EHF controls a distinct network of miRNAs with both oncogenic and tumor suppressor functions. Loss of ESE3/EHF resulted specifically in increased expression of miR-424, which has oncogenic properties in prostate epithelial cells. This represents a novel mechanism by which deregulation of ESE3/EHF impact on prostate tumorigenesis. Thus, targeting miR-424 could be a novel therapeutic strategy for prostate cancer. Citation Format: Cecilia Dallavalle, Domenico Albino, Gianluca Civenni, Paola Ostano, Davide Genini, Ramon Garcia-Escudero, Laura Curti, Sandra Pinton, Manuela Sarti, Giovanna Chiorino, Carlo V. Catapano, Giuseppina M. R. Carbone. MicroRNAs regulated by ESE3/EHF control important mediators of epithelial cell differentiation and stemness in prostate tumors. [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 1451. doi:10.1158/1538-7445.AM2014-1451