Genes In LNCaP Cells Research Articles

Stromal-epithelial interaction induces GALNT14 in prostate carcinoma cells.

Cell-cell communication is an important process in healthy tissue but also gains enhanced attention regarding pathological tissue. To date, the tumor microenvironment is gradually brought into focus when studying tumorigenesis. In the prostate gland, stromal and epithelial cells greatly interact to maintain homeostasis or tissue integrity. This study focuses on an indirect communication via soluble factors. To investigate the cell-cell interaction via soluble factors, the prostate carcinoma cell line LNCaP and the stromal primary cells p21 were co-cultured without direct contact and RNA was isolated at defined time points. Differences in gene expression were finally analyzed by RNA sequencing. RNA sequencing revealed a time-depending differential expression profile. Selected factors were subsequently characterized at molecular level and analyzed in human prostate tissue of different developmental stages as well as pathology. GALNT14 was one of the highest induced co-culture-specific genes in LNCaP cells. Detection in healthy tissue and BPH revealed an age-dependent decrease in GALNT14 expression. Moreover, in prostate carcinoma, GALNT14 expression heavily varied independent of the Gleason score. Overall, this work provides a basis for further studies related to paracrine stromal-epithelial interaction in prostate carcinoma and highlights the importance of GALNT14.

Prohibitin Links Cell Cycle, Motility and Invasion in Prostate Cancer Cells.

Prohibitin (PHB) is a tumour suppressor gene with several different molecular activities. PHB overexpression leads to G1/S-phase cell cycle arrest, and PHB represses the androgen receptor (AR) in prostate cancer cells. PHB interacts with and represses members of the E2F family in a manner that may also be AR-linked, therefore making the AR:PHB:E2F interaction axis highly complex. PHB siRNA increased the growth and metastatic potential of LNCaP mouse xenografts in vivo. Conversely, PHB ectopic cDNA overexpression affected several hundred genes in LNCaP cells. Furthermore, gene ontology analysis showed that in addition to cell cycle regulation, several members of the WNT family were significantly downregulated (WNT7B, WNT9A and WNT10B), as well as pathways for cell adhesion. Online GEO data studies showed PHB expression to be decreased in clinical cases of metastatic prostate cancer, and to be correlated with higher WNT expression in metastasis. PHB overexpression reduced prostate cancer cell migration and motility in wound-healing assays, reduced cell invasion through a Matrigel layer and reduced cellular attachment. In LNCaP cells, WNT7B, WNT9A and WNT10B expression were also upregulated by androgen treatment and downregulated by androgen antagonism, indicating a role for AR in the control of these WNT genes. However, these WNTs were strongly cell cycle regulated. E2F1 cDNA ectopic expression and PHB siRNA (both cell cycle promoting effects) increased WNT7B, WNT9A and WNT10B expression, and these genes were also upregulated as cells were released from G1 to S phase synchronisation, indicating further cell cycle regulation. Therefore, the repressive effects of PHB may inhibit AR, E2F and WNT expression and its loss may increase metastatic potential in human prostate cancer.

Fibronectin Modulates the Expression of miRNAs in Prostate Cancer Cell Lines.

Prostate cancer (PCa) is a significant cause of cancer-related deaths among men and companion animals, such as dogs. However, despite its high mortality and incidence rates, the molecular mechanisms underlying this disease remain to be fully elucidated. Among the many factors involved in prostate carcinogenesis, the extracellular matrix (ECM) plays a crucial role. This ECM in the prostate is composed mainly of collagen fibers, reticular fibers, elastic fibers, proteoglycans and glycoproteins, such as fibronectin. Fibronectin is a glycoprotein whose dysregulation has been implicated in the development of multiple types of cancer, and it has been associated with cell migration, invasion, and metastasis. Furthermore, our research group has previously shown that fibronectin induces transcriptional changes by modulating the expression of protein coding genes in LNCaP cells. However, potential changes at the post-transcriptional level are still not well understood. This study investigated the impact of exposure to fibronectin on the expression of a key class of regulatory RNAs, the microRNAs (miRNAs), in prostate cancer cell lines LNCaP and PC-3. Five mammalian miRNAs (miR-21, miR-29b, miR-125b, miR-221, and miR-222) were differentially expressed after fibronectin exposure in prostate cell lines. The expression profile of hundreds of mRNAs predicted to be targeted by these miRNAs was analyzed using publicly available RNA-Sequencing data (GSE64025, GSE68645, GSE29155). Also, protein-protein interaction networks and enrichment analysis were performed to gain insights into miRNA biological functions. Altogether, these functional analyzes revealed that fibronectin exposure impacts the expression of miRNAs potentially involved in PCa causing changes in critical signaling pathways such as PI3K-AKT, and response to cell division, death, proliferation, and migration. The relationship here demonstrated between fibronectin exposure and altered miRNA expression improves the comprehension of PCa in both men and other animals, such as dogs, which naturally develop prostate cancer.

Lysine Demethylase 6B Regulates Prostate Cancer Cell Proliferation by Controlling c-MYC Expression.

Elevated expression of lysine demethylase 6A (KDM6A) and lysine demethylase 6B (KDM6B) has been reported in prostate cancer (PCa). However, the mechanism underlying the specific role of KDM6A/B in PCa is still fragmentary. Here, we report novel KDM6A/B downstream targets involved in controlling PCa cell proliferation. KDM6A and KDM6B mRNAs were higher in prostate adenocarcinoma, lymph node metastatic site (LNCaP) but not in prostate adenocarcinoma, bone metastatic site (PC3) and prostate adenocarcinoma, brain metastatic site (DU145) cells. Higher KDM6A mRNA was confirmed at the protein level. A metastasis associated gene focused oligonucleotide array was performed to identify KDM6A/B dependent genes in LNCaP cells treated with a KDM6 family selective inhibitor, ethyl-3-(6-(4,5-dihydro-1H-benzo[d]azepin-3(2H)-yl)-2-(pyridin-2-yl)pyrimidin-4-ylamino)propanoate (GSK-J4). This identified five genes [V-myc myelocytomatosis viral oncogene homolog (avian) (c-MYC), neurofibromin 2 (merlin) (NF2), C-terminal binding protein 1 (CTBP1), EPH receptor B2 (EPHB2), and plasminogen activator urokinase receptor (PLAUR)] that were decreased more than 50% by GSK-J4, and c-MYC was the most downregulated gene. Array data were validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR), which detected a reduction in c-MYC steady state mRNA and prespliced mRNA, indicative of transcriptional repression of c-MYC gene expression. Furthermore, c-MYC protein was also decreased by GSK-J4. Importantly, GSK-J4 reduced mRNA and protein levels of c-MYC target gene, cyclinD1 (CCND1). Silencing of KDM6A/B with small interfering RNA (siRNA) confirmed that expression of both c-MYC and CCND1 are dependent on KDM6B. Phosphorylated retinoblastoma (pRb), a marker of G1 to S-phase transition, was decreased by GSK-J4 and KDM6B silencing. GSK-J4 treatment resulted in a decrease in cell proliferation and cell number, detected by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay and conventional cell counting, respectively. Consequently, we conclude that KDM6B controlling c-MYC, CCND1, and pRb contribute regulation of PCa cell proliferation, which represents KDM6B as a promising epigenetic target for the treatment of advanced PCa. SIGNIFICANCE STATEMENT: Lysine demethylase 6A (KDM6A) and 6B (KDM6B) were upregulated in prostate cancer (PCa). We reported novel KDM6A/B downstream targets controlling proliferation. Amongst 84 metastasis associated genes, V-myc myelocytomatosis viral oncogene homolog (avian) (c-MYC) was the most inhibited gene by KDM6 inhibitor, ethyl-3-(6-(4,5-dihydro-1H-benzo[d]azepin-3(2H)-yl)-2-(pyridin-2-yl)pyrimidin-4-ylamino)propanoate (GSK-J4). This was accompanied by decreased c-MYC targets, cyclinD1 (CCND1) and phosphorylated retinoblastoma (pRb), which were KDM6B dependent. GSK-J4 decreased proliferation and cell counting. We conclude that KDM6B controlling c-MYC, CCND1, and pRb contribute regulation of PCa proliferation.

Paxillin regulated genomic networks in prostate cancer

Paxillin is extensively involved in focal adhesion signaling and kinase signaling throughout the plasma membrane and cytoplasm. However, recent studies in prostate cancer suggest that paxillin also plays a critical role in regulating gene expression within the nucleus, serving as a liaison between cytoplasmic and nuclear MAPK and Androgen Receptor (AR) signaling. Here we used RNA-seq to examine the paxillin-regulated transcriptome in several human prostate cancer cell lines. First, we examined paxillin effects on androgen-mediated transcription in control or paxillin-depleted AR-positive LNCaP and C4-2 human prostate cancer cells. In androgen-dependent LNCaP cells, we found over 1000 paxillin-dependent androgen-responsive genes, some of which are involved in endocrine therapy resistance. Most paxillin-dependent AR-mediated genes in LNCaP cells were no longer paxillin-dependent in androgen-sensitive, castration-resistant C4-2 cells, suggesting that castration-resistance may markedly alter paxillin effects on genomic AR signaling. To examine the paxillin-regulated transcriptome in the absence of androgen signaling, we performed RNA-seq in AR-negative PC3 human prostate cancer cells. Paxillin enhanced several pro-proliferative pathways, including the CyclinD/Rb/E2F and DNA replication/repair pathways. Additionally, paxillin suppressed pro-apoptotic genes, including CASP1 and TNFSF10. Quantitative PCR confirmed that these pathways are similarly regulated by paxillin in LNCaP and C4-2 cells. Functional studies showed that, while paxillin stimulated cell proliferation, it had minimum effect on apoptosis. Thus, paxillin appears to be an important transcriptional regulator in prostate cancer, and analysis of its transcriptome might lead to novel approaches toward the diagnosis and treatment of this important disease.

Apoptosis‐Inducing Herbs for Prostate Cancer Prevention and Treatment

BackgroundProstate cancer is the most common non‐skin cancer in men. The conventional treatment regimen for prostate cancer is chemical castration, which has adverse effects, and tumors often recur following treatment. Therefore, developing an alternative therapeutic plan for prostate cancer is very much desirable. The purpose of this study is to identify and validate natural herbs that can induce apoptosis of prostate cancer cells.MethodsSaw Palmetto (Serenoa repens, I), Sanqi (Panax notoginsengI), and Ban Lan Gen (Isatis tinctoria) are extracted in alcohol at various concentrations. We performed HPLC to characterize these herbs, and then treated LNCaP prostate cancer cells with the ethanol extracts of these herbs. The viability of the treated and control cells was assessed by MTT Cell Proliferation Assay and flow cytometry. Real‐Time Quantitative PCR was performed to determine the expression levels of apoptotic genes.ResultsWe show here that Saw Palmetto (Serenoa repens, I), Sanqi (Panax notoginsengI), and Ban Lan Gen (Isatis tinctoria) have the corresponding standard HPLC molecular profiles. They decreased the cell viability and enhanced apoptosis of LNCaP cells based upon MTT assay and flow cytometric analysis. Our preliminary quantitative RT‐PCR data also showed the elevated the expression levels of apoptotic genes in LNCaP cells treated by the herb extracts.DiscussionThe data show that cell proliferation of prostate cancer cells is reduced by treatment in vitro of the herbs studied. Despite the limitations of this in vitro study, these data provide exciting evidence for the anti‐cancer properties of these herbs and could contribute to new treatments for prostate cancer.Support or Funding InformationThis work was funded by Grant 4 and 13 from Bastyr University.

Novel Nine-Exon AR Transcripts (Exon 1/Exon 1b/Exons 2-8) in Normal and Cancerous Breast and Prostate Cells.

Nearly 20 different transcripts of the human androgen receptor (AR) are reported with two currently listed as Refseq isoforms in the NCBI database. Isoform 1 encodes wild-type AR (type 1 AR) and isoform 2 encodes the variant AR45 (type 2 AR). Both variants contain eight exons: they share common exons 2–8 but differ in exon 1 with the canonical exon 1 in isoform 1 and the variant exon 1b in isoform 2. Splicing of exon 1 or exon 1b is reported to be mutually exclusive. In this study, we identified a novel exon 1b (1b/TAG) that contains an additional TAG trinucleotide upstream of exon 1b. Moreover, we identified AR transcripts in both normal and cancerous breast and prostate cells that contained either exon 1b or 1b/TAG spliced between the canonical exon 1 and exon 2, generating nine-exon AR transcripts that we have named isoforms 3a and 3b. The proteins encoded by these new AR variants could regulate androgen-responsive reporters in breast and prostate cancer cells under androgen-depleted conditions. Analysis of type 3 AR-GFP fusion proteins showed partial nuclear localization in PC3 cells under androgen-depleted conditions, supporting androgen-independent activation of the AR. Type 3 AR proteins inhibited androgen-induced growth of LNCaP cells. Microarray analysis identified a small set of type 3a AR target genes in LNCaP cells, including genes known to modulate growth and proliferation of prostate cancer (PCGEM1, PEG3, EPHA3, and EFNB2) or other types of human cancers (TOX3, ST8SIA4, and SLITRK3), and genes that are diagnostic/prognostic biomarkers of prostate cancer (GRINA3, and BCHE).

TBECH, 1,2-dibromo-4-(1,2 dibromoethyl) cyclohexane, alters androgen receptor regulation in response to mutations associated with prostate cancer

Point mutations in the AR ligand-binding domain (LBD) can result in altered AR structures leading to changes of ligand specificity and functions. AR mutations associated to prostate cancer (PCa) have been shown to result in receptor activation by non-androgenic substances and anti-androgenic drugs. Two AR mutations known to alter the function of anti-androgens are the ART877A mutation, which is frequently detected mutation in PCa tumors and the ARW741C that is rare and has been derived in vitro following exposure of cells to the anti-androgen bicalutamide. AR activation by non-androgenic environmental substances has been suggested to affect PCa progression. In the present study we investigated the effect of AR mutations (ARW741C and ART877A) on the transcriptional activation following exposure of cells to an androgenic brominated flame retardant, 1,2-dibromo-4-(1,2 dibromoethyl) cyclohexane (TBECH, also named DBE-DBCH). The AR mutations resulted in higher interaction energies and increased transcriptional activation in response to TBECH diastereomer exposures. The ART877A mutation rendered AR highly responsive to low levels of DHT and TBECH and led to increased AR nuclear translocation. Gene expression analysis showed a stronger induction of AR target genes in LNCaP cells (ART877A) compared to T-47D cells (ARWT) following TBECH exposure. Furthermore, AR knockdown experiments confirmed the AR dependency of these responses. The higher sensitivity of ART877A and ARW741C to low levels of TBECH suggests that cells with these AR mutations are more susceptible to androgenic endocrine disrupters.

Activin A regulates microRNAs and gene expression in LNCaP cells.

Prostate cancer (PCa) is an increasing health issue worldwide. For patients with advanced castration-resistant PCa (CRPC) treatment options are limited and overall survival is relatively short. Paired with this, non-invasive diagnostic options are yet to be established. Activins are members of the TGF-β superfamily and have been linked to prostate physiology. For instance, activin A is an inhibitor of growth in the prostate. A novel class of non-coding RNA, microRNAs (miRNAs) have been intrinsically linked to a range of cellular processes and carcinogenesis. No studies have investigated the impact of activin A on miRNA expression in PCa cell lines. Hence, the objective of this study was to determine the effect of activin A on miRNA expression and downstream target genes in PCa. Activin-sensitive (LNCaP) and insensitive (PC3) prostate cells were treated with 50 ng/ml of activin A for 72 hr. To examine miRNA expression following treatment, SYBR RT-qPCR miRNA arrays were used in conjunction with TaqMan RT-qPCR. MiRPath-TarBase analysis was conducted using the miRNAs that were significantly altered following activin A treatment of LNCaP cells to highlight enriched target genes within biological pathways. Highlighted target genes were assessed using pathway-focused TGF-β and cell cycle SYBR RT-qPCR arrays. Activin A treatment altered nine miRNAs in LNCaP cells: miR-222-3p, miR-15b-5p, miR-93-5p, miR-18a-5p, and let-7i-5p were significantly decreased, while miR-30a/30d-5p, let-7c, and miR-196b-5p were significantly increased versus media control. In PC3 cells five miRNAs were altered: miR-130a-3p, miR-7-5p, and miR-140-3p were significantly decreased while miR-191-5p and miR-26a-5p were significantly increased versus media control. MiRPath-TarBase analysis highlighted that the miRNAs significantly altered in LNCaP cells targeted genes contained in activin A-related KEGG pathways. Furthermore, when LNCaP cells were treated with activin A the expression of the targeted genes was the inverse of the expression of activin A-mediated miRNAs. This study demonstrated the ability of activin A to modulate miRNA expression in PCa cell lines and suggests a correlative relationship between miRNA expression and downstream target genes in LNCaP cells. This study provides impetus for further studies into activin A and miRNAs in PCa. Prostate 76:951-963, 2016. © 2016 Wiley Periodicals, Inc.

The AhR Ligand, TCDD, Regulates Androgen Receptor Activity Differently in Androgen-Sensitive versus Castration-Resistant Human Prostate Cancer Cells.

The reported biological effects of TCDD include induction of drug metabolizing enzymes, wasting syndrome and tumor promotion. TCDD elicits most of its effects through binding the aryl hydrocarbon receptor (AhR). TCDD induced degradation of AhR has been widely reported and requires ubiquitination of the protein. The rapid depletion of AhR following TCDD activation serves as a mechanism to modulate AhR mediated gene induction. In addition to inducing AhR degradation, TCDD has been reported to induce degradation of hormone receptors. The studies reported here, evaluate the effect of TCDD exposure on androgen receptor (AR) expression and activity in androgen-sensitive LNCaP and castration-resistant C4-2 prostate cancer cells. Our results show that TCDD exposure does not induce AhR or AR degradation in C4-2 cells. However, both AhR and AR are degraded in LNCaP cells following TCDD exposure. In addition, TCDD enhances AR phosphorylation and induces expression of AR responsive genes in LNCaP cells. Our data reveals that TCDD effect on AR expression and activity differs in androgen-sensitive and castration-resistant prostate cancer cell models.

Identification of novel genes that regulate androgen receptor signaling and growth of androgen-deprived prostate cancer cells.

Prostate cancer progression to castration refractory disease is associated with anomalous transcriptional activity of the androgen receptor (AR) in an androgen-depleted milieu. To identify novel gene products whose downregulation transactivates AR in prostate cancer cells, we performed a screen of enzymatically-generated shRNA lenti-libraries selecting for transduced LNCaP cells with elevated expression of a fluorescent reporter gene under the control of an AR-responsive promoter. The shRNAs present in selected populations were analyzed using high-throughput sequencing to identify target genes. Highly enriched gene targets were then validated with siRNAs against selected genes, testing first for increased expression of luciferase from an AR-responsive promoter and then for altered expression of endogenous androgen-regulated genes in LNCaP cells. We identified 20 human genes whose silencing affected the expression of exogenous and endogenous androgen-responsive genes in prostate cancer cells grown in androgen-depleted medium. Knockdown of four of these genes upregulated the expression of endogenous AR targets and siRNAs targeting two of these genes (IGSF8 and RTN1) enabled androgen-independent proliferation of androgen-dependent cells. The effects of IGSF8 appear to be mediated through its interaction with a tetraspanin protein, CD9, previously implicated in prostate cancer progression. Remarkably, homozygous deletions of IGSF8 are found almost exclusively in prostate cancers but not in other cancer types. Our study shows that androgen independence can be achieved through the inhibition of specific genes and reveals a novel set of genes that regulate AR signaling in prostate cancers.

Genome-wide analysis of androgen receptor binding sites in prostate cancer cells.

The transformation of prostate cancer from an androgen-dependent state to an androgen-independent state is a lethal progression. Alterations in transcriptional programs are the basis of prostate cancer deterioration. The androgen receptor (AR), a member of the nuclear hormone receptor superfamily, mediates prostate cancer progression by functioning primarily through the ligand-activated transcription of target genes. Therefore, a detailed map of AR-regulated genes and AR genomic binding sites is required for hormone-naive and castration-resistant prostate cancer. Through the use of chromatin immunoprecipitation in combination with direct sequencing, 4,143 AR binding sites were defined in the LNCaP androgen-sensitive prostate cancer cell line. Using the same method, 2,380 AR binding regions were identified in the LNCaP-AI long-term androgen-deprived cell line. Approximately 8.5% (354/4,143) of the binding regions were mapped to within 2 kb of the transcription start site (TSS) in the LNCaP cells, while ∼12.6% (299/2,380) were mapped to within 2 kb of the TSS in the LNCaP-AI cells. In total, the study mapped 2,796 genes in LNCaP cells and 1,854 genes in LNCaP-AI cells. The cell lines shared 789 mutual genes. In addition, gene ontology (GO) analysis of the genes revealed that there was a notable overlap between the GO terms in the LNCaP cells and LNCaP-AI cells. However, GO terms within the biological process domain that were only observed in the LNCaP-AI cells included the reproduction process, death, immune system process, multi-organism process, pigmentation and viral reproduction. The major genes in the different GO terms were TNFAIP8, RTN4, APP and SYNE1. Through analyzing the AR binding sites in the two cell types, the present study aimed to map potential AR-regulated genes, identify their associated transcription factors and provide a new perspective on the biological processes in the development of prostate cancer. The results provided a valuable data set that furthered the understanding of the genome-wide analysis of AR binding sites in prostate cancer cells, which may be exploited for the development of novel prostate cancer therapeutic strategies.

Osteopontin-c mediates the upregulation of androgen responsive genes in LNCaP cells through PI3K/Akt and androgen receptor signaling.

Androgen receptor (AR) signaling is a key pathway modulating prostate cancer (PCa) progression. Several steps in this pathway have been investigated in order to propose novel treatment strategies for advanced PCa. Total osteopontin (OPN) has been described as a biomarker for PCa, in addition to its role in activating the progression of this tumor. Based on the known effects of the OPNc splice variant on PCa progression, the present study investigated whether this isoform can also modulate AR signaling. In order to test this, an in vitro model was used in which LNCaP cells were cultured in the presence of conditioned medium (CM) secreted by PCa cells overexpressing OPNc (OPNc-CM). The activation of AR signaling was evaluated by measuring the expression levels of AR-responsive genes (ARGs) using quantitative polymerase chain reaction and specific oligonucleotides. The data demonstrated that all nine tested ARGs (Fgf8, TMPRSS2, Greb1, Cdk2, Ndrg1, Cdk1, Pmepa1, Psa and Ar) are significantly upregulated in response to OPNc-CM compared with LNCaP cells cultured in CM secreted by control cells transfected with empty expression vector. The specific involvement of OPNc was demonstrated by depleting OPNc from OPNc-CM using an anti-OPNc neutralizing antibody. In addition, by using a phosphoinositide 3-kinase (PI3K)-specific inhibitor and AR antagonists, such as flutamide and bicalutamide, it was also observed that upregulation of ARGs in response to OPNc-CM involves PI3K signaling and depends on the AR. In conclusion, these data indicated that OPNc is able to activate AR signaling through the PI3K pathway and the AR. These data further corroborate our previous data, revealing the OPNc splice variant to be a key molecule that is able to modulate key signaling pathways involved in PCa progression.

Determinants of Gli2 co-activation of wildtype and naturally truncated androgen receptors.

Gli2, a transcription factor in the Hedgehog pathway, is overexpressed in castrate-resistant prostate cancer (PCa). Previously we showed that Gli2 overexpression increased transcriptional activity of androgen receptor (AR) and conferred androgen growth-independence to normally growth-dependent PCa cells. Here we localized the regions of AR-Gli2 protein interaction and determined the domains within Gli2 needed for AR co-activation. Co-immunoprecipitation and GST-pulldown assays were used to define AR-Gli binding domains. Co-activation assays using androgen-responsive promoter reporters were used to define Gli2 regions needed for AR co-activation. Chromatin immunoprecipitation (ChIP) assays were used to confirm nuclear interactions of Gli2 with AR in PCa cells. The Gli2 C-terminal domain (CTD) is sufficient for AR co-activation. Two elements within the CTD were required: (1) an AR binding domain within aa628-897; and (2) at least part of the Gli2 transactivation domain within aa1252-1586. In turn, Gli2 binds the tau5/AF5 ligand-independent activation domain in the AR N-terminus. Mutations in the WxxLF motif in tau5/AF5 greatly diminished binding to Gli2-CTD. Gli2 interaction with AR tau5/AF5 was further substantiated by the ability of Gli2/Gli2-CTD to co-activate truncated AR splice variants (AR-V7/ARV567es). ChIP assays confirmed that Gli2 associates with chromatin at androgen response elements found near androgen-responsive genes in LNCaP cells. These assays also showed that AR associates with chromatin containing a Gli-response element near a Gli-responsive gene. Our findings indicate that Gli2 overexpression in PCa cells might support development of castration resistant PCa through AR co-activation and suggests that AR might modulate transcription from Gli2.

Paracrine interactions between LNCaP prostate cancer cells and bioengineered bone in 3D in vitro culture reflect molecular changes during bone metastasis

As microenvironmental factors such as three-dimensionality and cell–matrix interactions are increasingly being acknowledged by cancer biologists, more complex 3D in vitro models are being developed to study tumorigenesis and cancer progression. To better understand the pathophysiology of bone metastasis, we have established and validated a 3D indirect co-culture model to investigate the paracrine interactions between prostate cancer (PCa) cells and human osteoblasts. Co-culture of the human PCa, LNCaP cells embedded within polyethylene glycol hydrogels with human osteoblasts in the form of a tissue engineered bone construct (TEB), resulted in reduced proliferation of LNCaP cells. LNCaP cells in both monoculture and co-culture were responsive to the androgen analog, R1881, as indicated by an increase in the expression (mRNA and/or protein induction) of androgen-regulated genes including prostate specific antigen and fatty acid synthase. Microarray gene expression analysis further revealed an up-regulation of bone markers and other genes associated with skeletal and vasculature development and a significant activation of transforming growth factor β1 downstream genes in LNCaP cells after co-culture with TEB. LNCaP cells co-cultured with TEB also unexpectedly showed similar changes in classical androgen-responsive genes under androgen-deprived conditions not seen in LNCaP monocultures. The molecular changes of LNCaP cells after co-culturing with TEBs suggest that osteoblasts exert a paracrine effect that may promote osteomimicry and modulate the expression of androgen-responsive genes in LNCaP cells. Taken together, we have presented a novel 3D in vitro model that allows the study of cellular and molecular changes occurring in PCa cells and osteoblasts that are relevant to metastatic colonization of bone. This unique in vitro model could also facilitate cancer biologists to dissect specific biological hypotheses via extensive genomic or proteomic assessments to further our understanding of the PCa-bone crosstalk.

Labda-8(17),12,14-trien-19-oic Acid Contained in Fruits ofCupressus sempervirensSuppresses Benign Prostatic Hyperplasia in Rat andIn VitroHuman Models Through Inhibition of Androgen and STAT-3 Signaling

Fruit extract of Cupressus sempervirens (CS), which is used traditionally to treat Benign Prostatic Hyperplasia (BPH)-like urinary symptoms in patients, was scientifically validated for anti-BPH activity. The ethanolic fruit extract of CS inhibited proliferation of human BPH-stromal cells and the activity was localized to its chloroform-soluble, diterpene-rich fraction. Eight major diterpenes isolated from this fraction exhibited moderate to potent activity and the most active diterpene (labda-8(17),12,14-trien-19-oic acid) exhibited an IC50 of 37.5 μM (antiproliferative activity against human BPH-stromal cells). It significantly inhibited activation (phosphorylation) of Stat-3 in BPH-stromal cells and prevented transactivation of androgen sensitive KLK3/PSA and TMPRSS2 genes in LNCaP cells. Labda-8(17),12,14-trien-19-oic acid-rich CS fraction prevented prostatic hyperplasia in rat model and caused TUNEL labeling of stromal cells with lower expressions of IGF-I, TGF-ß and PCNA, and bcl-2/bax ratio. Human BPH tissues exhibited precise lowering of stromal component after incubation in labda-8(17),12,14-trien-19-oic acid, ex vivo. We conclude that labda-8(17),12,14-trien-19-oic acid contained in CS exhibits anti-BPH activity through inhibition of stromal proliferation and suppression of androgen action in the prostate, presenting a unique lead structure for further optimization of anti-BPH activity.

Peroxisome Proliferation-Activated Receptor δ Agonist GW0742 Interacts Weakly with Multiple Nuclear Receptors, Including the Vitamin D Receptor

A high-throughput screening campaign was conducted to identify small molecules with the ability to inhibit the interaction between the vitamin D receptor (VDR) and steroid receptor coactivator 2. These inhibitors represent novel molecular probes for modulating gene regulation mediated by VDR. Peroxisome proliferator-activated receptor (PPAR) δ agonist GW0742 was among the identified VDR-coactivator inhibitors and has been characterized herein as a pan nuclear receptor antagonist at concentrations of > 12.1 μM. The highest antagonist activity for GW0742 was found for VDR and the androgen receptor. Surprisingly, GW0742 behaved as a PPAR agonist and antagonist, activating transcription at lower concentrations and inhibiting this effect at higher concentrations. A unique spectroscopic property of GW0742 was identified as well. In the presence of rhodamine-derived molecules, GW0742 increased the fluorescence intensity and level of fluorescence polarization at an excitation wavelength of 595 nm and an emission wavelength of 615 nm in a dose-dependent manner. The GW0742-inhibited NR-coactivator binding resulted in a reduced level of expression of five different NR target genes in LNCaP cells in the presence of agonist. Especially VDR target genes CYP24A1, IGFBP-3, and TRPV6 were negatively regulated by GW0742. GW0742 is the first VDR ligand inhibitor lacking the secosteroid structure of VDR ligand antagonists. Nevertheless, the VDR-meditated downstream process of cell differentiation was antagonized by GW0742 in HL-60 cells that were pretreated with the endogenous VDR agonist 1,25-dihydroxyvitamin D3.

Abstract B19: Induction of apoptosis and antitumor activity of the AKT inhibitor AZD5363 is enhanced by combination with hormonal therapies in preclinical models of prostate cancer

Abstract Activation of AKT by mechanisms including loss of the tumor suppressors PTEN and INPP4B is very common in prostate cancer, and has been correlated with poor prognosis and progression to androgen independent disease. Recently, a reciprocal feedback mechanism has been shown to occur between the androgen and AKT signalling axes in prostate cancer models. AZD5363 monotherapy induced apoptosis and inhibited growth of PTEN null, AR+ prostate cancer cell lines including LNCaP, C4-2 and a flutamide resistant variant of PC346C (GI50 10 – 100 nM), and showed moderate anti-proliferative activity in AR amplified, PTEN wild type VCaP cells (GI50 ∼ 1 μM). The PTEN wild type, AR negative DU-145, BPH1 and RWPE1 cell lines were relatively resistant. Monotherapy AZD5363 also caused dosedependent growth inhibition of the PTEN null, AR+ HID28 prostate cancer explant model in vivo. Treatment with AZD5363 increased expression of a panel of androgen regulated genes in LNCaP cells, whereas the same panel of genes was down-regulated when AZD5363 was combined with the novel anti-androgen MDV-3100. Combination of AZD5363 with MDV-3100 resulted in greater inhibition of growth and induction of apoptosis in vitro than monotherapy treatment with these agents, indicating a synergistic interaction. Moreover, combination of AZD5363 and bicalutamide resulted in greater efficacy than AZD5363 monotherapy in LNCaP xenografts in vivo. The data show that whilst AZD5363 has considerable monotherapy activity in AR+/PTEN null prostate cancer models, the combination with anti-androgens merits further evaluation as a possible means to achieve greater anti-tumor activity. AZD5363 is currently in Phase 1 clinical trials. Citation Format: Claire Crafter, Barry R. Davies, Christian Thomas, Francois Lamoureux, Hannah Greenwood, Juan M. Garcia-Martinez, Lorraine Mooney, Sabina Cosulich, Martin Gleave, Amina Zoubeidi. Induction of apoptosis and antitumor activity of the AKT inhibitor AZD5363 is enhanced by combination with hormonal therapies in preclinical models of prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B19.

Abstract A49: Characterization of pre-B-cell leukemia transcription factor 1 in prostate cancer

Abstract The main purpose of our project is to determine the role of Pre-B Cell Leukemia Transcription Factor 1 (PBX1) in prostate cancer development and progression. In this study of PBX1, we used immunohistochemical staining of benign and malignant prostate tissue as well as immunoblotting and Real-Time RT-PCR of different prostate cancer cell lines to characterize the expression pattern and regulation of PBX1. Furthermore, we transfected LNCaP with short interference RNA (siRNA) against PBX1 to determine its role in proliferation and differentiation of prostate cancer cells. Our analyses of PBX1 expression in prostate cancer cell lines indicate that the level of PBX1 is higher in LNCaP and VCaP compared to LNCaP C4-2B and DU145, at both the mRNA and protein levels. Immunohistochemical analysis of PBX1 showed that the protein is mainly expressed in basal cells of benign tissue and in a minor sub-fraction of cells located in the luminal compartment. Tumor tissue lacks basal cells and the expression level of PBX1 was therefore down-regulated in malignant compared to benign tissue. In less differentiated tumor areas, the frequency of PBX1 positive cells was lower than in highly differentiated tumors. Thus, an inverse correlation between PBX1 staining and Gleason grade was observed. The TMAstudy included 42 patients radically operated for prostate cancer in 2004 (108 spots) and no significant correlations between PBX1 expression and clinical parameters were observed. Cyclic AMP has previously been shown to induce growth arrest and neurite outgrowth of prostate cancer cells. We have shown that the level of PBX1 mRNA is down-regulated by cAMP. Transient transfection of PBX1 siRNA in LNCaP cells mimicked the morphological changes observed when LNCaP cells are stimulated with cAMP (forskolin) and both treatments reduced the growth rate of LNCaP cells. Several cell cycle regulated genes have been identified as cAMP target genes in LNCaP cells and we are currently investigating whether these are PBX target genes. Furthermore, preliminary studies indicate decreased expression of luminal differentiation markers in LNCaP cells transfected with PBX1 siRNA. Based on these preliminary studies, we suggest that PBX1 might play a role in regulation of development and/or progression of prostate cancer, as suggested for other types of cancers, like ovarian and renal cancers. Citation Format: Hakon Ramberg, Ayham Alshbib, Viktor Berge, Wang Wanzhong, Aud Svindland, Kristin Austlid Taskén. Characterization of pre-B-cell leukemia transcription factor 1 in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A49.

Stearoyl-CoA desaturase induces lipogenic gene expression in prostate cancer cells and inhibits ceramide-induced cell death

Perturbation of metabolism with increased expression of lipogenic enzymes is a common characteristic of human cancers, including prostate cancer. In the present work the overexpression of stearoyl-CoA desaturase (SCD) in LNCaP cells led to increased mRNA levels of fatty acid synthase (FAS) and acetyl-CoA-carboxylase-α, whereas micro RNA-mediated silencing of SCD inhibited the expression of these lipogenic genes in LNCaP cells. Treatment with the FAS-specific inhibitor cerulenin inhibited SCD induction of LNCaP cell proliferation. In addition, a transient transfection assay revealed the capability of cerulenin to suppress SCD and dihydrotestosterone induction of androgen receptor transcriptional activity. Furthermore, overexpression of SCD in LNCaP cells produced marked resistance to ceramide-induced cell death with reduced poly(ADP-ribose) polymerase (PARP) cleavage. In contrast, silencing of SCD expression increased Bax protein in LNCaP cells. Furthermore, addition of ceramide to SCD knockdown LNCaP cells increased cell death and caspase-3 activity with drastic increase of PARP cleavage. Together, the data indicate that SCD may provide resistance of prostate cancer cells to ceramide-induced cell death.