Repeated trans-arterial treatments of LDL-DHA nanoparticles induce multiple pathways of tumor cell death in hepatocellular carcinoma bearing rats.

Introduction: Repeated hepatic arterial delivery of therapeutic agents to the liver by percutaneously implanted port-catheter systems has been widely used to treat unresectable liver cancer. This approach is applied to assess the therapeutic efficacy of repeated low-density lipoprotein-docosahexaenoic acid (LDL-DHA) nanoparticle treatments in a rat model of hepatocellular carcinoma. Methods: N1S1 hepatoma bearing rats underwent placement of a percutaneously implanted hepatic artery port-catheter system and were allocated to untreated, control LDL-triolein (LDL-TO) or LDL-DHA nanoparticle infusions groups. Treatments were performed every three days over a nine day study period. MRI was performed at baseline and throughout the study. At the end of the study tissue samples were collected for analyses. Results and Discussion: Implantation of the port catheters was successful in all rats. MRI showed that repeated infusions of LDL-DHA nanoparticles significantly impaired the growth of the rat hepatomas eventually leading to tumor regression. The tumors in the LDL-TO treated group showed delayed growth, while the untreated tumors grew steadily throughout the study. Histopathology and MRI support these findings demonstrating extensive tumor necrosis in LDL-DHA treated groups while the control groups displayed minor necrosis. Molecular and biochemical analyses also revealed that LDL-DHA treated tumors had increased levels of nuclear factor-kappa B and lipid peroxidation and depletion of glutathione peroxidase 4 relative to the control groups. Evidence of both ferroptosis and apoptosis tumor cell death was observed following LDL-DHA treatments. In conclusion repeated transarterial infusions of LDL-DHA nanoparticles provides sustained repression of tumor growth in a rat hepatoma model.

The helix-loop-helix transcriptional regulator Id4 is required for terminal differentiation of luminal epithelial cells in the prostate.

Inhibitor of differentiation 4 (Id4), a member of the helix-loop-helix family of transcriptional regulators has emerged as a tumor suppressor in prostate cancer. In this study we investigated the effect of loss of Id4 (Id4-/-) on mouse prostate development. Histological analysis was performed on prostates from 25 days, 3 months and 6 months old Id4-/- mice. Expression of Amacr, Ck8, Ck18, Fkbp51, Fkbp52, androgen receptor, Pten, sca-1 and Nkx3.1 was investigated by immunohistochemistry. Results were compared to the prostates from Nkx3.1-/- mice. Id4-/- mice had smaller prostates with fewer and smaller tubules. Subtle PIN like lesions were observed at 6mo. Decreased Nkx3.1 and Pten and increased stem cell marker sca-1, PIN marker Amacr and basal cell marker p63 was observed at all ages. Persistent Ck8 and Ck18 expression suggested that loss of Id4 results in epithelial commitment but not terminal differentiation in spite of active Ar. Loss of Id4 attenuates normal prostate development and promotes hyperplasia/ dysplasia with PIN like lesions. The results suggest that loss of Id4 maintains stem cell phenotype of "luminal committed basal cells", identifying a unique prostate developmental pathway regulated by Id4.

Spermatogonial Gene Networks Selectively Couple to Glutathione and Pentose Phosphate Metabolism but Not Cysteine Biosynthesis.

In adult males, spermatogonia maintain lifelong spermatozoa production for oocyte fertilization. To understand spermatogonial metabolism we compared gene profiles in rat spermatogonia to publicly available mouse, monkey, and human spermatogonial gene profiles. Interestingly, rat spermatogonia expressed metabolic control factors Foxa1, Foxa2, and Foxa3. Germline Foxa2 was enriched in Gfra1Hi and Gfra1Low undifferentiated A-single spermatogonia. Foxa2-bound loci in spermatogonial chromatin were overrepresented by conserved stemness genes (Dusp6, Gfra1, Etv5, Rest, Nanos2, Foxp1) that intersect bioinformatically with conserved glutathione/pentose phosphate metabolism genes (Tkt, Gss, Gc l c , Gc l m, Gpx1, Gpx4, Fth), marking elevated spermatogonial GSH:GSSG. Cystine-uptake and intracellular conversion to cysteine typically couple glutathione biosynthesis to pentose phosphate metabolism. Rat spermatogonia, curiously, displayed poor germline stem cell viability in cystine-containing media, and, like primate spermatogonia, exhibited reduced transsulfuration pathway markers. Exogenous cysteine, cysteine-like mercaptans, somatic testis cells, and ferroptosis inhibitors counteracted the cysteine-starvation-induced spermatogonial death and stimulated spermatogonial growth factor activity in vitro.

Assessing the safety of transarterial locoregional delivery of low-density lipoprotein docosahexaenoic acid nanoparticles to the rat liver.

Hepatic-arterial infusion (HAI) of low-density lipoprotein (LDL) nanoparticles reconstituted with docosahexaenoic acid (DHA) (LDL-DHA) has been shown in a rat hepatoma model to be a promising treatment for hepatocellular carcinoma. To date, little is known regarding the safety of HAI of LDL-DHA to the liver. Therefore, we aimed to investigate the deposition, metabolism and safety of HAI of LDL-DHA (2, 4 or 8 mg/kg) in the rat. Following HAI, fluorescent labeled LDL nanoparticles displayed a biexponential plasma concentration time curve as the particles were rapidly extracted by the liver. Overall, increasing doses of HAI of LDL-DHA was well tolerated in the rat. Body weight, plasma biochemistry and histology were all unremarkable and molecular markers of inflammation did not increase with treatment. Lipidomics analyses showed that LDL-DHA was preferentially oxidized to the anti-inflammatory mediator, protectin DX. We conclude that HAI of LDL-DHA nanoparticles is not only safe, but provides potential hepatoprotective benefits.

An implanted port-catheter system for repeated hepatic arterial infusion of low-density lipoprotein-docosahexaenoic acid nanoparticles in normal rats: A safety study.

BACKGROUND: In recent years, small animal arterial port-catheter systems have been implemented in rodents with reasonable success. The aim of the current study is to employ the small animal port-catheter system to evaluate the safety of multiple hepatic-artery infusions (HAI) of low-density lipoprotein-docosahexaenoic acid (LDL-DHA) nanoparticles to the rat liver. METHODS: Wistar rats underwent surgical placement of indwelling HAI ports. Repeated administrations of PBS or LDL-DHA nanoparticles were performed through the port at baseline and days 3 and 6. Rats were sacrificed on day 9 at which point blood and various organs were collected for histopathology and biochemical analyses. RESULTS: The port-catheter systems were implanted successfully and repeated infusions of PBS or LDL-DHA nanoparticles were tolerated well by all animals over the duration of the study. Measurements of serum liver/renal function tests, glucose and lipid levels did not differ between control and LDL-DHA treated rats. The liver histology was unremarkable in the LDL-DHA treated rats and the expression of hepatic inflammatory regulators (NF-κß, IL-6 and CRP) were similar to control rats. Repeated infusions of LDL-DHA nanoparticles did not alter liver glutathione content or the lipid profile in the treated rats. The DHA extracted by the liver was preferentially metabolized to the anti-inflammatory DHA-derived mediator, protectin DX. CONCLUSION: Our findings indicate that repeated HAI of LDL-DHA nanoparticles is not only well tolerated and safe in the rat, but may also be protective to the liver.

Management of Hsp90-Dependent Protein Folding by Small Molecules Targeting the Aha1 Co-Chaperone.

Hsp90 plays an important role in health and is a therapeutic target for managing misfolding disease. Compounds that disrupt co-chaperone delivery of clients to Hsp90 target a subset of Hsp90 activities, thereby minimizing the toxicity of pan-Hsp90 inhibitors. Here, we have identified SEW04784 as a first-in-class inhibitor of the Aha1-stimulated Hsp90 ATPase activity without inhibiting basal Hsp90 ATPase. Nuclear magnetic resonance analysis reveals that SEW84 binds to the C-terminal domain of Aha1 to weaken its asymmetric binding to Hsp90. Consistent with this observation, SEW84 blocks Aha1-dependent Hsp90 chaperoning activities, including the in vitro and in vivo refolding of firefly luciferase, and the transcriptional activity of the androgen receptor in cell-based models of prostate cancer and promotes the clearance of phosphorylated tau in cellular and tissue models of neurodegenerative tauopathy. We propose that SEW84 provides a novel lead scaffold for developing therapeutic approaches to treat proteostatic disease.

Lipoprotein Drug Delivery Vehicles for Cancer: Rationale and Reason.

Lipoproteins are a family of naturally occurring macromolecular complexes consisting amphiphilic apoproteins, phospholipids, and neutral lipids. The physiological role of mammalian plasma lipoproteins is to transport their apolar cargo (primarily cholesterol and triglyceride) to their respective destinations through a highly organized ligand-receptor recognition system. Current day synthetic nanoparticle delivery systems attempt to accomplish this task; however, many only manage to achieve limited results. In recent years, many research labs have employed the use of lipoprotein or lipoprotein-like carriers to transport imaging agents or drugs to tumors. The purpose of this review is to highlight the pharmacologic, clinical, and molecular evidence for utilizing lipoprotein-based formulations and discuss their scientific rationale. To accomplish this task, evidence of dynamic drug interactions with circulating plasma lipoproteins are presented. This is followed by epidemiologic and molecular data describing the association between cholesterol and cancer.

A Small-Molecule Pan-Id Antagonist Inhibits Pathologic Ocular Neovascularization.

Id helix-loop-helix (HLH) proteins (Id1-4) bind E protein bHLH transcription factors, preventing them from forming active transcription complexes that drive changes in cell states. Id proteins are primarily expressed during development to inhibit differentiation, but they become re-expressed in adult tissues in diseases of the vasculature and cancer. We show that the genetic loss of Id1/Id3 reduces ocular neovascularization in mouse models of wet age-related macular degeneration (AMD) and retinopathy of prematurity (ROP). An in silico screen identifies AGX51, a small-molecule Id antagonist. AGX51 inhibits the Id1-E47 interaction, leading to ubiquitin-mediated degradation of Ids, cell growth arrest, and reduced viability. AGX51 is well-tolerated in mice and phenocopies the genetic loss of Id expression in AMD and ROP models by inhibiting retinal neovascularization. Thus, AGX51 is a first-in-class compound that antagonizes an interaction formerly considered undruggable and that may have utility in the management of multiple diseases.

Inactivation of ID4 promotes a CRPC phenotype with constitutive AR activation through FKBP52.

Castration-resistant prostate cancer (CRPC) is the emergence of prostate cancer cells that have adapted to the androgen-depleted environment of the prostate. In recent years, targeting multiple chaperones and co-chaperones (e.g., Hsp27, FKBP52) that promote androgen receptor (AR) signaling and/or novel AR regulatory mechanisms have emerged as promising alternative treatments for CRPC. We have shown that inactivation of inhibitor of differentiation 4 (ID4), a dominant-negative helix loop helix protein, promotes de novo steroidogenesis and CRPC with a gene expression signature that resembles constitutive AR activity in castrated mice. In this study, we investigated the underlying mechanism through which loss of ID4 potentiates AR signaling. Proteomic analysis between prostate cancer cell line LNCaP (L+ns) and LNCaP lacking ID4 (L(-)ID4) revealed elevated levels of Hsp27 and FKBP52, suggesting a role for these AR-associated co-chaperones in promoting constitutively active AR signaling in L(-)ID4 cells. Interestingly, protein interaction studies demonstrated a direct interaction between ID4 and the 52-kDa FK506-binding protein (FKBP52) in vitro, but not with AR. An increase in FKBP52-dependent AR transcriptional activity was observed in L(-)ID4 cells. Moreover, pharmacological inhibition of FKBP52-AR signaling, by treatment with MJC13, attenuated the tumor growth, weight, and volume in L(-)ID4 xenografts. Together, our results demonstrate that ID4 selectively regulates AR activity through direct interaction with FKBP52, and its loss, promotes CRPC through FKBP52-mediated AR signaling.

ID4 regulates transcriptional activity of wild type and mutant p53 via K373 acetylation.

Given that mutated p53 (50% of all human cancers) is over-expressed in many cancers, restoration of mutant p53 to its wild type biological function has been sought after as cancer therapy. The conformational flexibility has allowed to restore the normal biological function of mutant p53 by short peptides and small molecule compounds. Recently, studies have focused on physiological mechanisms such as acetylation of lysine residues to rescue the wild type activity of mutant p53. Using p53 null prostate cancer cell line we show that ID4 dependent acetylation promotes mutant p53 DNA-binding capabilities to its wild type consensus sequence, thus regulating p53-dependent target genes leading to subsequent cell cycle arrest and apoptosis. Specifically, by using wild type, mutant (P223L, V274F, R175H, R273H), acetylation mimics (K320Q and K373Q) and non-acetylation mimics (K320R and K373R) of p53, we identify that ID4 promotes acetylation of K373 and to a lesser extent K320, in turn restoring p53-dependent biological activities. Together, our data provides a molecular understanding of ID4 dependent acetylation that suggests a strategy of enhancing p53 acetylation at sites K373 and K320 that may serve as a viable mechanism of physiological restoration of mutant p53 to its wild type biological function.

Intra-tumoral delivery of functional ID4 protein via PCL/maltodextrin nano-particle inhibits prostate cancer growth.

ID4, a helix loop helix transcriptional regulator has emerged as a tumor suppressor in prostate cancer. Epigenetic silencing of ID4 promotes prostate cancer whereas ectopic expression in prostate cancer cell lines blocks cancer phenotype. To directly investigate the anti-tumor property, full length human recombinant ID4 encapsulated in biodegradable Polycaprolactone/Maltodextrin (PCL-MD) nano-carrier was delivered to LNCaP cells in which the native ID4 was stably silenced (LNCaP(-)ID4). The cellular uptake of ID4 resulted in increased apoptosis, decreased proliferation and colony formation. Intratumoral delivery of PCL-MD ID4 into growing LNCaP(-)ID4 tumors in SCID mice significantly reduced the tumor volume compared to the tumors treated with chemotherapeutic Docetaxel. The study supports the feasibility of using nano-carrier encapsulated ID4 protein as a therapeutic. Mechanistically, ID4 may assimilate multiple regulatory pathways for example epigenetic re-programming, integration of multiple AR co-regulators or signaling pathways resulting in tumor suppressor activity of ID4.

ID4 promotes AR expression and blocks tumorigenicity of PC3 prostate cancer cells.

Deregulation of tumor suppressor genes is associated with tumorigenesis and the development of cancer. In prostate cancer, ID4 is epigenetically silenced and acts as a tumor suppressor. In normal prostate epithelial cells, ID4 collaborates with androgen receptor (AR) and p53 to exert its tumor suppressor activity. Previous studies have shown that ID4 promotes tumor suppressive function of AR whereas loss of ID4 results in tumor promoter activity of AR. Previous study from our lab showed that ectopic ID4 expression in DU145 attenuates proliferation and promotes AR expression suggesting that ID4 dependent AR activity is tumor suppressive. In this study, we examined the effect of ectopic expression of ID4 on highly malignant prostate cancer cell, PC3. Here we show that stable overexpression of ID4 in PC3 cells leads to increased apoptosis and decreased cell proliferation and migration. In addition, in vivo studies showed a decrease in tumor size and volume of ID4 overexpressing PC3 cells, in nude mice. At the molecular level, these changes were associated with increased androgen receptor (AR), p21, and AR dependent FKBP51 expression. At the mechanistic level, ID4 may regulate the expression or function of AR through specific but yet unknown AR co-regulators that may determine the final outcome of AR function.

A Modified Coupled Spectrophotometric Method to Detect 2-5 Oligoadenylate Synthetase Activity in Prostate Cell Lines.

BACKGROUND: 2'-5' oligoadenylate synthetases (OAS) are interferon inducible enzymes that polymerizes ATP to 2'-5'-linked oligomers of adenylate (2-5As). As part of the innate immune response, these enzymes are activated by viral double stranded RNA or mRNAs with significant double stranded structure. The 2-5As in turn activate RNaseL that degrade single stranded RNAs. Three distinct forms of OAS exist in human cells (OAS1, 2 and 3) with each form having multiple spliced variants. The OAS enzymes and their spliced variants have different enzyme activities. OAS enzymes also play a significant role in regulating multiple cellular processes such as proliferation and apoptosis. Moreover, Single nucleotide polymorphisms that alter OAS activity are also associated with viral infection, diabetes and cancer. Thus detection of OAS enzyme activity with a simple spectrophotometric method in cells will be important in clinical research. RESULTS: Here we propose a modified coupled spectrophotometric assay to detect 2-5 oligoadenylate synthetase (OAS) enzyme activity in prostate cell lines as a model system. The OAS enzyme from prostate cancer cell lysates was purified using Polyinosinic: polycytidylic acid (poly I:C) bound activated sepharose beads. The activated OAS enzyme eluted from Sepharose beads showed expression of p46 isoform of OAS1, generally considered the most abundant OAS isoform in elutes from DU14 cell line but not in other prostate cell line. In this assay the phosphates generated by the OAS enzymatic reaction is coupled with conversion of the substrate 2-amino-6-mercapto-7-methylpurine ribonucleoside (methylthioguanosine, a guanosine analogue; MESG) to a purine base product, 2-amino-6-mercapto-7-methylpurine and ribose1-phosphate via a catalyst purine nucleoside phosphorylase (phosphorylase) using a commercially available pyrophosphate kit. The absorbance of the purine base product is measured at 360 nm. The higher levels of phosphates detected in DU145 cell line indicates more activity of OAS in this prostate cancer cell line. CONCLUSION: The modified simple method detected OAS enzyme activity with sensitivity and specificity, which could help in detection of OAS enzymes avoiding the laborious and radioactive methods.

Relative Stability of Wild-Type and Mutant p53 Core Domain: A Molecular Dynamic Study.

The p53 protein is a stress response protein that functions primarily as a tetrameric transcription factor. A tumor suppressor p53 binds to a specific DNA sequence and transactivates target genes, leading to cell cycle apoptosis. Encoded by the human gene TP53, p53 is a stress response protein that functions primarily as a tetrameric transcription factor. This gene regulates a large number of genes in response to a variety of cellular functions, including oncogene activation and DNA damage. Mutations in p53 are common in human cancer types. Herein we mutate a wild-type p53, 1TSR with four of its mutated proteins. The energy for the wild-type and mutated proteins is calculated by using molecular dynamics simulations along with simulated annealing. Our results show significant differences in energy between hotspot mutations and the wild type. Based on the findings, we investigate the correlation between molar masses of the target residue and the relative energy with respect to the wild type. Our results indicate that the relative energy changes play a pivotal role in bioactivity, in conformity with observations in the rate of mutation in biology.

NRG1 and KITL Signal Downstream of Retinoic Acid in the Germline to Support Soma-Free Syncytial Growth of Differentiating Spermatogonia.

Defined culture systems supporting spermatogonial differentiation will provide experimental platforms to study spermatogenesis. However, germline-intrinsic signaling mechanisms sufficient to support spermatogonial differentiation without somatic cells remain largely undefined. Here, we analyzed EGF superfamily receptor and ligand diversity in rat testis cells, and delineated germline-intrinsic signaling via an ERBB3 co-transducer, ERBB2, as essential for retinoic acid-induced syncytial growth by differentiating spermatogonia. Like the ERBB2/3 agonist NRG1, we found KIT Ligand (KITL) robustly supported spermatogonial differentiation without serum or somatic cells. ERBB2 inhibitors failed to disrupt KITL-dependent spermatogonial development, and, KITL prevented ERBB3-deficient spermatogonial degeneration upon differentiation. Thus, we report NRG1 and KITL activate alternative pathways downstream of retinoic acid signaling in the germline that are essential for stem cells to undergo pre-meiotic steps of spermatogenesis in culture. Robust serum/soma-free spermatogonial differentiation opens new doors to study mammalian germ cell biology in culture, which will facilitate the discovery of spermatogenic factors that can drive meiotic progression in vitro.

Polycaprolactone/maltodextrin nanocarrier for intracellular drug delivery: formulation, uptake mechanism, internalization kinetics, and subcellular localization.

Prostate cancer (PCa) disease progression is associated with significant changes in intracellular and extracellular proteins, intracellular signaling mechanism, and cancer cell phenotype. These changes may have direct impact on the cellular interactions with nanocarriers; hence, there is the need for a much-detailed understanding, as nanocarrier cellular internalization and intracellular sorting mechanism correlate directly with bioavailability and clinical efficacy. In this study, we report the differences in the rate and mechanism of cellular internalization of a biocompatible polycaprolactone (PCL)/maltodextrin (MD) nanocarrier system for intracellular drug delivery in LNCaP, PC3, and DU145 PCa cell lines. PCL/MD nanocarriers were designed and characterized. PCL/MD nanocarriers significantly increased the intracellular concentration of coumarin-6 and fluorescein isothiocyanate-labeled bovine serum albumin, a model hydrophobic and large molecule, respectively. Fluorescence microscopy and flow cytometry analysis revealed rapid internalization of the nanocarrier. The extent of nanocarrier cellular internalization correlated directly with cell line aggressiveness. PCL/MD internalization was highest in PC3 followed by DU145 and LNCaP, respectively. Uptake in all PCa cell lines was metabolically dependent. Extraction of endogenous cholesterol by methyl-ß-cyclodextrin reduced uptake by 75%±4.53% in PC3, 64%±6.01% in LNCaP, and 50%±4.50% in DU145, indicating the involvement of endogenous cholesterol in cellular internalization. Internalization of the nanocarrier in LNCaP was mediated mainly by macropinocytosis and clathrin-independent pathways, while internalization in PC3 and DU145 involved clathrin-mediated endocytosis, clathrin-independent pathways, and macropinocytosis. Fluorescence microscopy showed a very diffused and non-compartmentalized subcellular localization of the PCL/MD nanocarriers with possible intranuclear localization and minor colocalization in the lysosomes with time.

Inhibitor of differentiation 4 (ID4) acts as an inhibitor of ID-1, -2 and -3 and promotes basic helix loop helix (bHLH) E47 DNA binding and transcriptional activity.

The four known ID proteins (ID1-4, Inhibitor of Differentiation) share a homologous helix loop helix (HLH) domain and act as dominant negative regulators of basic-HLH transcription factors. ID proteins also interact with many non-bHLH proteins in complex networks. The expression of ID proteins is increasingly observed in many cancers. Whereas ID-1, ID-2 and ID-3, are generally considered as tumor promoters, ID4 on the contrary has emerged as a tumor suppressor. In this study we demonstrate that ID4 heterodimerizes with ID-1, -2 and -3 and promote bHLH DNA binding, essentially acting as an inhibitor of inhibitors of differentiation proteins. Interaction of ID4 was observed with ID1, ID2 and ID3 that was dependent on intact HLH domain of ID4. Interaction with bHLH protein E47 required almost 3 fold higher concentration of ID4 as compared to ID1. Furthermore, inhibition of E47 DNA binding by ID1 was restored by ID4 in an EMSA binding assay. ID4 and ID1 were also colocalized in prostate cancer cell line LNCaP. The alpha helix forming alanine stretch N-terminal, unique to HLH ID4 domain was required for optimum interaction. Ectopic expression of ID4 in DU145 prostate cancer line promoted E47 dependent expression of CDKNI p21. Thus counteracting the biological activities of ID-1, -2 and -3 by forming inactive heterodimers appears to be a novel mechanism of action of ID4. These results could have far reaching consequences in developing strategies to target ID proteins for cancer therapy and understanding biologically relevant ID-interactions.

Targeted Germline Modifications in Rats Using CRISPR/Cas9 and Spermatogonial Stem Cells.

Organisms with targeted genomic modifications are efficiently produced by gene editing in embryos using CRISPR/Cas9 RNA-guided DNA endonuclease. Here, to facilitate germline editing in rats, we used CRISPR/Cas9 to catalyze targeted genomic mutations in rat spermatogonial stem cell cultures. CRISPR/Cas9-modified spermatogonia regenerated spermatogenesis and displayed long-term sperm-forming potential following transplantation into rat testes. Targeted germline mutations in Epsti1 and Erbb3 were vertically transmitted from recipients to exclusively generate "pure," non-mosaic mutant progeny. Epsti1 mutant rats were produced with or without genetic selection of donor spermatogonia. Monoclonal enrichment of Erbb3 null germlines unmasked recessive spermatogenesis defects in culture that were buffered in recipients, yielding mutant progeny isogenic at targeted alleles. Thus, spermatogonial gene editing with CRISPR/Cas9 provided a platform for generating targeted germline mutations in rats and for studying spermatogenesis.

Basic helix loop helix (bHLH) transcription factor 3 (TCF3, E2A) is regulated by androgens in prostate cancer cells.

TCF3 (E2A) is a multifunctional basic helix loop helix (bHLH) transcription factor that is over-expressed in prostate cancer (PCa) as compared to normal prostate and that it acts as a tumor promoter in PCa. Given the diverse biological pathways regulated/influenced by TCF3, little is known about the mechanisms that regulate its expression. TCF3 expression in androgen sensitive LNCaP and insensitive C81 PCa cell lines was determined following treatments with androgen receptor (AR) agonist R1881 and antagonist Casodex. In silico analysis was used to discover putative Androgen Response Elements (ARE) in the TCF3 promoter/intron region. Chromatin Immunoprecipitation (ChIP) with AR antibody and luciferase reporter assays on the above mentioned cell lines was used to confirm AR biding and AR dependent transcriptional activity respectively. The results were confirmed by demonstrating TCF3 expression in LNCaP PCa xenograft models. The results suggested that TCF3 transcript increased in response to R1881 in LNCaP cells but was constitutively expressed in C-81 cell lines. The promoter/Intron region of the TCF3 gene was predicted to contain two putative ARE sites ARE1 and ARE2. ChIP after treatment of LNCaP and C81 cells with R1881 and Casodex showed that the ARE1 and ARE2 were bound by AR in LNCaP cells only in the presence of R1881, whereas C81 cells showed constitutive AR binding. Similar results were observed in luciferase reporter assays indicating that TCF3 is activated by AR in LNCaP cell lines whereas it is independent of androgens in C81 cell line. Luciferase reporter assays also confirmed that ARE1 alone drives androgen dependent transcription. TCF3 expression was only observed in castration resistant LNCaP xenografts in castrated mice. In conclusion, we demonstrate that in PCa androgen receptor regulates the expression of TCF3 which is mediated in part via a consensus androgen response element. The shift in TCF3 expression from androgen regulated to androgen independent during prostate cancer progression, together with lack of expression in normal prostate may provide mechanistic basis underlying the transition of androgen receptor from a tumor suppressor to an oncogene in prostate cancer.

Prostate cancer epigenome.

Prostate cancer is a major health burden within the ever-increasingly aging US population. The molecular mechanisms involved in prostate cancer are diverse and heterogeneous. In this context, epigenetic changes, both global and gene specific, are now an emerging alternate mechanism in disease initiation and progression. The three major risk factors in prostate cancer: age, geographic ancestry, and environment are all influenced by epigenetics and additional significant insight is required to gain an understanding of the underlying mechanisms. The androgen receptor and its downstream effector pathways, central to prostate cancer initiation and progression, are subject to a multitude of epigenetic alterations. In this review we focus on the global perspective of epigenetics and the use of recent next-generation sequencing platforms to interrogate epigenetic changes in the prostate cancer genome.