Foxm1 expression in prostate epithelial cells is essential for prostate carcinogenesis.

The treatment of advanced prostate cancer (PCa) remains a challenge. Identification of new molecular mechanisms that regulate PCa initiation and progression would provide targets for the development of new cancer treatments. The Foxm1 transcription factor is highly up-regulated in tumor cells, inflammatory cells, and cells of tumor microenvironment. However, its functions in different cell populations of PCa lesions are unknown. To determine the role of Foxm1 in tumor cells during PCa development, we generated two novel transgenic mouse models, one exhibiting Foxm1 gain-of-function and one exhibiting Foxm1 loss-of-function under control of the prostate epithelial-specific Probasin promoter. In the transgenic adenocarcinoma mouse prostate (TRAMP) model of PCa that uses SV40 large T antigen to induce PCa, loss of Foxm1 decreased tumor growth and metastasis. Decreased prostate tumorigenesis was associated with a decrease in tumor cell proliferation and the down-regulation of genes critical for cell proliferation and tumor metastasis, including Cdc25b, Cyclin B1, Plk-1, Lox, and Versican. In addition, tumor-associated angiogenesis was decreased, coinciding with reduced Vegf-A expression. The mRNA and protein levels of 11ß-Hsd2, an enzyme playing an important role in tumor cell proliferation, were down-regulated in Foxm1-deficient PCa tumors in vivo and in Foxm1-depleted TRAMP C2 cells in vitro. Foxm1 bound to, and increased transcriptional activity of, the mouse 11ß-Hsd2 promoter through the -892/-879 region, indicating that 11ß-Hsd2 was a direct transcriptional target of Foxm1. Without TRAMP, overexpression of Foxm1 either alone or in combination with inhibition of a p19(ARF) tumor suppressor caused a robust epithelial hyperplasia, but was insufficient to induce progression from hyperplasia to PCa. Foxm1 expression in prostate epithelial cells is critical for prostate carcinogenesis, suggesting that inhibition of Foxm1 is a promising therapeutic approach for prostate cancer chemotherapy.

Small extravesicular microRNA in head and neck squamous cell carcinoma and its potential as a liquid biopsy for early detection.

BACKGROUND: The objective was to assess secretion of small extracellular vesicular microRNA (exo-miRNA) in head and neck squamous cell carcinoma (HNSCC) according to human papillomavirus (HPV) status, and determine the translational potential as a liquid biopsy for early detection. METHODS: This study employed a combination of cell culture and case-control study design using archival pretreatment serum. Small extracellular vesicles (sEV) were isolated from conditioned culture media and human serum samples via differential ultracentrifugation. miRNA-sequencing was performed on each sEV isolate. RESULTS: There were clear exo-miRNA profiles that distinguished HNSCC cell lines from nonpathologic oral epithelial control cells. While there was some overlap among profiles across all samples, there were apparent differences in exo-miRNA profiles according to HPV-status. Importantly, differential exo-miRNA profiles were also apparent in serum from early-stage HNSCC cases relative to cancer-free controls. CONCLUSIONS: Our findings indicate that exo-miRNA are highly dysregulated in HNSCC and support the potential of exo-miRNA as biomarkers for HNSCC.

Estrogen-like disruptive effects of dietary exposure to bisphenol A or 17α-ethinyl estradiol in CD1 mice.

Bisphenol A (BPA) is an endocrine disrupting chemical that is ubiquitous in wild and built environments. Due to variability in study design, the disruptive effects of BPA have proven difficult to experimentally replicate. This study was designed to assess the disruptive actions of dietary BPA exposure, while carefully controlling for known confounders. Parental CD1 mice were acclimated to defined diet containing BPA (0.03, 0.3, 3, 30, or 300 ppm) or 17α-ethinyl estradiol (EE; 0.0001, 0.001, and 0.01 ppm) and bred to produce progeny (F1) that were maintained through adulthood on the same diet as the parents. In F1 females, uterine weights were increased in all EE and the 30-ppm BPA-exposure groups, demonstrating model sensitivity and estrogen-like actions of BPA. In BPA-exposed females, no treatment-related differences were observed in parental reproductive function, or in the timing of puberty and metabolic function in female offspring. In F1 males, modest changes in body weight, adiposity and glucose tolerance, consistent with improved metabolic function, were observed. Associated with increased prolactin and increased circulating testosterone levels, balanopreputial separation was accelerated by 0.03 and 3.0 ppm BPA and anogenital distance at postnatal day 21 was increased in males by 0.03 ppm BPA. Sperm counts were also increased with 3.0 ppm BPA exposures. Overall, BPA was found to have modest, sex specific endocrine disruptive effects on a variety of end points below the established no observed adverse effect level. The dose response characteristics for many of the effects were nonmonotonic and not predictable from high-dose extrapolations.

The androgen receptor inhibits transcription of GPER1 by preventing Sp1 and Sp3 from binding to the promoters in prostate cancer cells.

G-1, a GPER1 agonist, was shown to inhibit the growth of castration-resistant mouse xenografts but not their parental androgen-dependent tumors. It is currently unknown how the androgen receptor (AR) represses GPER1 expression. Here, we found that two GPER1 mRNA variants (GPER1v2 and GPER1v4) were transcriptionally repressed, not via transcript destabilization, by the androgen-activated AR. Although no AR binding was found in all active promoters near GPER1, data from promoter assays suggested that both variants' promoters were inhibited by androgen treatment. Site-directed mutagenesis on Sp1/Sp3 binding sites revealed their role in supporting the basal expression of GPER1. Knockdown of Sp1 and Sp3 together but not separately repressed GPER1 expression whereas overexpression of both Sp1 and Sp3 together was required to alleviate AR repression of GPER1. Based on the chromatin immunoprecipitation data, Sp3 was found to bind to the promoters prior to the binding of Sp1 and RNA polymerase II. However, the binding of all three transcription factors was inhibited by DHT treatment. Concordantly, DHT treatment induced nuclear interactions between AR and Sp1 or Sp3. Taken together, these results indicate that AR represses transcription of GPER1 by binding to Sp1 and Sp3 independently to prevent their transactivation of the GPER1 promoters.

Characterization of cis elements of the probasin promoter necessary for prostate-specific gene expression.

BACKGROUND: The androgen-regulated probasin (PB) promoter has been used extensively to target transgenes to the prostate in transgenic mice; however, limited data exist on the mechanism that dictates prostate-specific gene expression. Tissue-specific gene expression involves synergistic effects among transcription factors associated in a complex bound to cis-acting DNA elements. METHODS: Using comprehensive linker scan mutagenesis, enzyme mobility shift and supershift assays, chromatin immunoprecipitation, and transgenic animal studies, we have extensively characterized the prostate-specific PB promoter. RESULTS: We identified a series of nonreceptor transcription factors that are bound to the prostate-specific rat PB promoter. These factors include several ubiquitously distributed proteins known to participate in steroid receptor-mediated transcription. In addition, we identified two tissue-specific DNA elements that are crucial in directing prostate-specific PB expression, and confirmed the functional importance of both elements in transgenic animal studies. These two elements are functionally interchangeable and can be bound by multiple protein complexes, including the forkhead transcription factor FoxA1, a "pioneer factor" that has a restricted distribution to some cells type that are ectoderm and endoderm in origin. Using transgenic mice, we further demonstrate that the minimal PB promoter region (-244/-96 bp) that encompasses these tissue-specific elements results in prostate-specific gene expression in transgenic mice, contains androgen receptor and FoxA1-binding sites, as well as ubiquitous transcription factor binding sites. CONCLUSION: We propose that these sequence-specific DNA-binding proteins, including tissue-restricted and ubiquitous factors, create the first level of transcriptional control, which responds to intracellular pathways that directs prostate-specific gene expression.

Monitoring mouse prostate development by profiling and imaging mass spectrometry.

Mass spectrometry-based tissue profiling and imaging are technologies that allow identification and visualization of protein signals directly on thin sections cut from fresh frozen tissue specimens. These technologies were utilized to evaluate protein expression profiles in the normal mouse prostate during development (1-5 weeks of age), at sexual maturation (6 weeks of age), and in adult prostate (at 10, 15, or 40 weeks of age). The evolution of protein expression during normal prostate development and maturation were subsequently compared with 15-week prostate tumors derived from genetically engineered mice carrying the Large T antigen gene under regulation of the prostate-specific probasin promoter (LPB-Tag mouse model for prostate cancer). This approach identified proteins differentially expressed at specific time points during prostate development. Furthermore expression of some of these proteins, for example probasin and spermine-binding protein, were associated with prostate maturation, and prostate tumor formation resulted in their loss of expression. Cyclophilin A, a protein found in other cancers, was differentially alpha-acetylated on the N terminus, and both isoforms appeared during normal prostate and prostate tumor development. Imaging mass spectrometry localized the protein signals to specific prostatic lobes or regions. Thus, tissue profiling and imaging can be utilized to analyze the ontogeny of protein expression during prostate morphogenesis and tumorigenesis and identify proteins that could potentially serve as biomarkers for prostate cancer.

Identification of Differential Patterns of Oxidative Biomarkers in Prostate Cancer Progression.

INTRODUCTION: Oxidative stress has been found to be associated with the progression of prostate cancer (PCa); however, human studies which identify differential roles of each oxidation pathway in PCa progression are lacking. We aimed to identify which oxidative stress markers, specifically lipid and global oxidation and glycation, are associated with PCa progression. PATIENTS AND METHODS: We recruited 3 groups of patients from a urologic clinic at the University of Cincinnati Medical Center: men with PCa who had undergone prostatectomy, men with PCa under watchful waiting, and men with benign prostatic hyperplasia (BPH). We used the most commonly used lipid oxidation marker, F2-isoprostanes; global oxidation markers, fluorescent oxidation products (FlOPs); and the commonly used marker for advanced glycation end products, carboxymethyllysine. These biomarkers were measured in plasma samples at baseline entry. Plasma prostate-specific antigen (PSA) was measured at enrollment and follow-up visits. RESULTS: Compared with men with BPH, men with PCa who had undergone prostatectomy had 26% (P = .01) higher levels of F2-isoprostanes and 20% (P = .08) higher levels of carboxymethyllysine. All the oxidation markers were similar when comparing men under watchful waiting with men with BPH. When examining the associations between baseline oxidation markers and follow-up PSAs, we found that different oxidation markers had differential patterns associated with PSA elevation. F2-isoprostanes were positively associated with PSA elevation among men with PCa; FlOP_320 was positively associated with PSA elevation among both men with PCa and men with BPH, whereas among men with PCa under watchful waiting, FlOP_360 and FlOP_400 had opposite trends of associations with PSA elevation. CONCLUSIONS: Our study suggested that high levels of lipid oxidation were associated with PCa progression, whereas different global oxidation markers had different patterns associated with PCa progression. Large-scale clinical studies are needed to confirm our associations. Our study provides a comprehensive view of the relationship between biomarkers and PCa progression.

Exposure to perfluorooctanoic acid (PFOA) decreases neutrophil migration response to injury in zebrafish embryos.

OBJECTIVE: Perfluorooctanoic acid (PFOA) is a ubiquitous environmental contaminant and a known immune suppressant in humans and experimental animal models. Studies on PFOA have focused on suppression of the adaptive immune response; however, little is known of the impact on innate immunity, especially during embryogenesis. Therefore, we utilized the zebrafish chemotaxis assay coupled with in situ hybridization for myeloperoxidase expression to determine the effects of PFOA exposure on neutrophil migration in the developing zebrafish embryo. Zebrafish embryos are a well-established in vivo model that exhibit high homology with the development of human innate immunity. RESULTS: Treatment of zebrafish with increasing concentrations of PFOA identified the lethal concentration in 50% of the embryos (LC50) to be 300 mg/L. Utilizing the zebrafish chemotaxis assay, this study showed that wounding induced significant neutrophil migration to the site of injury, and that neutrophil number in the wound region was significantly reduced in response to 48-h PFOA exposure (well below doses causing acute mortality). This study demonstrates that the developing embryo is sensitive to PFOA exposure and that PFOA can modify the innate immune system during embryonic development. These results lay the groundwork for future investigation on the mechanisms underlying PFOA-induced developmental immunotoxicity.

Comparability of the small RNA secretome across human biofluids concomitantly collected from healthy adults.

Small extracellular vesicles (sEV) are nano-sized (40-150 nm), membrane-encapsulated vesicles that are released by essentially all cells into the extracellular space and function as intercellular signaling vectors through the horizontal transfer of biologic molecules, including microRNA (miRNA) and other small non-coding RNA (ncRNA), that can alter the phenotype of recipient cells. sEV are present in essentially all extracellular biofluids, including serum, urine and saliva, and offer a new avenue for discovery and development of novel biomarkers of various disease states and exposures. The objective of this study was to systematically interrogate similarities and differences between sEV ncRNA derived from saliva, serum and urine, as well as cell-free small ncRNA (cf-ncRNA) from serum. Saliva, urine and serum were concomitantly collected from 4 healthy donors to mitigate potential bias that can stem from interpersonal and temporal variability. sEV were isolated from each respective biofluid, along with cf-RNA from serum. sEV were isolated from the respective biofluids via differential ultracentrifugation with a 30% sucrose cushion to minimize protein contamination. Small RNA-sequencing was performed on each sample, and cluster analysis was performed based on ncRNA profiles. While some similarities existed in terms of sEV ncRNA cargo across biofluids, there are also notable differences in ncRNA class and ncRNA secretion, with sEV in each biofluid bearing a unique ncRNA profile, including major differences in composition by ncRNA class. We conclude that sEV ncRNA cargo varies according to biofluid, so thus should be carefully selected and interpreted when designing or contrasting translational or epidemiological studies.

Identification, characterization, and biological relevance of prostate cancer stem cells from clinical specimens.

Cancer stem cells (CSCs) are a reservoir of tumor cells that exhibit the properties of self-renewal and the ability to re-establish the heterogeneous cell population of a tumor. They appear therapy-resistant and may be the underlying cause of recurrent disease. Using prostate as a model, this review presents the CSC hypothesis and discusses the role of the androgen receptor in CSCs, the methods used for isolating CSCs, and the therapeutic challenges CSCs have for cancer therapy.

Prostate cancer cells with stem cell characteristics reconstitute the original human tumor in vivo.

Cancer may arise from a cancer stem/progenitor cell that shares characteristics with its normal counterpart. We report the reconstitution of the original human prostate cancer specimen from epithelial cell lines (termed HPET for human prostate epithelial/hTERT) derived from this sample. These tumors can be described in terms of Gleason score, a classification not applied to any of the transgenic mouse models currently developed to mimic human disease. Immunohistochemical and Western blot analyses indicate that they do not express androgen receptor or p63, similar to that reported for prostate stem cells. These cell lines also express embryonic stem markers (Oct4, Nanog, and Sox2) as well as early progenitor cell markers (CD44 and Nestin) in vitro. Clonally derived HPET cells reconstitute the original human tumor in vivo and differentiate into the three prostate epithelial cell lineages, indicating that they arise from a common stem/progenitor cell. Serial transplantation experiments reconstitute the tumors, suggesting that a fraction of parental or clonally derived HPET cells have self-renewal potential. Thus, this model may enhance our understanding of human tumor development and provide a mechanism for studying cancer stem/progenitor cells in differentiation, tumorigenesis, preclinical testing, and the development of drug resistance.

DJ-1 binds androgen receptor directly and mediates its activity in hormonally treated prostate cancer cells.

The oncogene DJ-1 has been associated with multiple cancers, including prostate cancer, where it can be stabilized by androgens and antiandrogens. However, little data exist on the expression pattern and function of DJ-1 in prostate cancer. To address the function of DJ-1 in prostate, a yeast two-hybrid screen was done to identify novel DJ-1 binding proteins. The androgen receptor (AR) was identified and confirmed as a DJ-1 binding partner. This is the first evidence that DJ-1 directly interacts with AR. We also show that modulation of DJ-1 expression regulated AR transcriptional activity. Importantly, both the subcellular localization of DJ-1 and the interaction with AR are regulated by androgens and antiandrogens. Additionally, immunohistochemical staining on two human prostate cancer tissue arrays was done providing the first large-scale expression analysis of DJ-1 in prostate. DJ-1 expression did not change with Gleason pattern but increased after androgen deprivation therapy, indicating that it may be involved in the development of androgen independence. These data provide a novel mechanism where DJ-1-mediated regulation of AR may promote the progression of prostate cancer to androgen independence.

Constant Degradation of the Androgen Receptor by MDM2 Conserves Prostate Cancer Stem Cell Integrity.

Prostate cancer stem cells (CSC) are implicated in tumor initiation, cancer progression, metastasis, and the development of therapeutic-resistant disease. It is well known that the bulk of prostate cancer cells express androgen receptor (AR) and that androgens are required for prostate cancer growth, progression, and emergence of castration-resistant disease. In contrast, the small subpopulation of self-renewing CSCs exhibits an AR-negative (AR-) signature. The mechanisms underlying the absence of AR are unknown. Using CSC-like cell models isolated from clinical biopsy tissues, we identify the E3 ligase MDM2 as a key regulator of prostate CSC integrity. First, unlike what has been reported for the bulk of AR+ tumor cells where MDM2 regulates the temporal expression of AR during transcriptional activity, MDM2 in CSCs promoted the constant ubiquitination and degradation of AR, resulting in sustained loss of total AR protein. Second, MDM2 promoted CSC self-renewal, the expression of stem cell factors, and CSC proliferation. Loss of MDM2 reversed these processes and induced expression of full-length AR (and not AR variants), terminal differentiation into luminal cells, and cell death. Selectively blocking MDM2-mediated activity in combination with androgen/AR-targeted therapy may offer a novel strategy for eliminating AR- CSCs in addition to the bulk of AR+ prostate cancer cells, decreasing metastatic tumor burden and inhibiting the emergence of therapeutic resistance.Significance: These findings provide a novel mechanistic aspect of prostate cancer cell stemness that advances our understanding of the diverse transcriptional activity that bypasses AR in contributing to therapeutic resistance, tumor progression, and metastasis.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/6/1124/F1.large.jpg.

Comprehensive mapping of the methylation landscape of 16 CpG-dense regions in oral and pharyngeal squamous cell carcinoma.

Aim: The goal of this study was to comprehensively interrogate and map DNA methylation across 16 CpG-dense regions previously associated with oral and pharyngeal squamous cell carcinoma (OPSCC). Materials & methods: Targeted multiplex bisulfite amplicon sequencing was performed on four OPSCC cell lines and primary non-neoplastic oral epithelial cells. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed for a subset of associated genes. Results: There was clear differential methylation between one or more OPSCC cell lines and control cells for the majority of CpG-dense regions. Conclusion: Targeted multiplex bisulfite amplicon sequencing allowed us to efficiently map methylation across the entire region of interest with a high degree of sensitivity and helps shed light on novel differentially methylated regions that may have value as biomarkers of OPSCC.

Differential Actions of Estrogen Receptor α and ß via Nongenomic Signaling in Human Prostate Stem and Progenitor Cells.

Human prostate stem and progenitor cells express estrogen receptor (ER)α and ERß and exhibit proliferative responses to estrogens. In this study, membrane-initiated estrogen signaling was interrogated in human prostate stem/progenitor cells enriched from primary epithelial cultures and stem-like cell lines from benign and cancerous prostates. Subcellular fractionation and proximity ligation assays localized ERα and ERß to the cell membrane with caveolin-1 interactions. Exposure to 17ß-estradiol (E2) for 15 to 60 minutes led to sequential phosphorylation of signaling molecules in MAPK and AKT pathways, IGF1 receptor, epidermal growth factor receptor, and ERα, thus documenting an intact membrane signalosome that activates diverse downstream cascades. Treatment with an E2-dendrimer conjugate or ICI 182,870 validated E2-mediated actions through membrane ERs. Overexpression and knockdown of ERα or ERß in stem/progenitor cells identified pathway selectivity; ERα preferentially activated AKT, whereas ERß selectively activated MAPK cascades. Furthermore, prostate cancer stem-like cells expressed only ERß, and brief E2 exposure activated MAPK but not AKT cascades. A gene subset selectively regulated by nongenomic E2 signaling was identified in normal prostate progenitor cells that includes BGN, FOSB, FOXQ1, and MAF. Membrane-initiated E2 signaling rapidly modified histone methyltransferases, with MLL1 cleavage observed downstream of phosphorylated AKT and EZH2 phosphorylation downstream of MAPK signaling, which may jointly modify histones to permit rapid gene transcription. Taken together, the present findings document ERα and ERß membrane-initiated signaling in normal and cancerous human prostate stem/progenitor cells with differential engagement of downstream effectors. These signaling pathways influence normal prostate stem/progenitor cell homeostasis and provide novel therapeutic sites to target the elusive prostate cancer stem cell population.

Stem cells: The root of prostate cancer?

The cancer stem cell (CSC) model states that tumors contain a reservoir of self-renewing cells that maintain the heterogeneous cell population of the tumor. These cells appear to be resistant to therapy and can therefore survive to repopulate the tumor during progression to therapy resistant disease. The biology of CSCs is still not definitive since it is difficult to isolate them from solid tumors and analyze their characteristics in vitro. Another challenge is to correlate these characteristics with tumor development and progression in vivo. Using the prostate CSC as a model, this review presents the CSC hypothesis, reviews the origin, identification and functions of prostate CSCs, and discusses the clinical implications and therapeutic challenges CSCs have for cancer therapy.

Hedgehog pathway activity in the LADY prostate tumor model.

BACKGROUND: Robust Hedgehog (Hh) signaling has been implicated as a common feature of human prostate cancer and an important stimulus of tumor growth. The role of Hh signaling has been studied in several xenograft tumor models, however, the role of Hh in tumor development in a transgenic prostate cancer model has never been examined. RESULTS: We analyzed expression of Hh pathway components and conserved Hh target genes along with progenitor cell markers and selected markers of epithelial differentiation during tumor development in the LADY transgenic mouse model. Tumor development was associated with a selective increase in Ihh expression. In contrast Shh expression was decreased. Expression of the Hh target Patched (Ptc) was significantly decreased while Gli1 expression was not significantly altered. A survey of other relevant genes revealed significant increases in expression of Notch-1 and Nestin together with decreased expression of HNF3a/FoxA1, NPDC-1 and probasin. CONCLUSION: Our study shows no evidence for a generalized increase in Hh signaling during tumor development in the LADY mouse. It does reveal a selective increase in Ihh expression that is associated with increased expression of progenitor cell markers and decreased expression of terminal differentiation markers. These data suggest that Ihh expression may be a feature of a progenitor cell population that is involved in tumor development.

Comprehensive microRNA-sequencing of exosomes derived from head and neck carcinoma cells in vitro reveals common secretion profiles and potential utility as salivary biomarkers.

Exosomes are nano-scale, membrane encapsulated vesicles that are released by cells into the extracellular space and function as intercellular signaling vectors through horizontal transfer of biologic molecules, including microRNA (miRNA). There is evidence that cancer-derived exosomes enable the tumor to manipulate its microenvironment, thus contributing to the capacity of the tumor for immune evasion, growth, invasion, and metastatic spread. The objective of this study was to characterize differential secretion of exosomal miRNA by head and neck squamous cell carcinoma (HNSCC) and identify a set of candidate biomarkers that could be detected in non-invasive saliva samples. We isolated exosomes from conditioned media from 4 HNSCC cell lines and oral epithelial control cells and applied miRNA-sequencing to comprehensively characterize their miRNA cargo and compare transcript levels of each HNSCC cell line to that of oral epithelial control cells. A candidate set of miRNA differentially secreted by all 4 HNSCC cell lines was further evaluated in saliva collected from HNSCC patients and healthy controls. We observed extensive differences in exosomal miRNA content between HNSCC cells when compared to normal oral epithelial control cells, with a high degree of overlap in exosomal miRNA profiles between the 4 distinct HNSCC cell lines. Importantly, several of the exosomal miRNA secreted solely by cancer cells in culture were detected at substantially elevated levels in saliva from HNSCC patients relative to saliva from healthy controls. These findings provide important insight into tumor biology and yields a promising set of candidate HNSCC biomarkers for use with non-invasive saliva samples.

Mechanisms leading to the development of hormone-resistant prostate cancer.

Advanced and metastatic prostate cancers remain potentially lethal tumors. Although androgen deprivation therapy remains the most effective treatment, patients who progress to androgen independence die of their disease. This article focuses on the mechanisms by which hormone resistance develops, including the reactivation of androgen receptor during androgen deprivation therapy, the role of cancer stem cells, and the emergence of epithelial-mesenchymal transition cells, which have increased metastatic potential. It is through an enhanced understanding of these mechanisms that new therapies can be developed to combat this disease.

NE-10 neuroendocrine cancer promotes the LNCaP xenograft growth in castrated mice.

Increases in neuroendocrine (NE) cells and their secretory products are closely correlated with tumor progression and androgen-independent prostate cancer. However, the mechanisms by which NE cells influence prostate cancer growth and progression, especially after androgen ablation therapy, are poorly understood. To investigate the role of NE cells on prostate cancer growth, LNCaP xenograft tumors were implanted into nude mice. After the LNCaP tumors were established, the NE mouse prostate allograft (NE-10) was implanted on the opposite flank of these nude mice to test whether NE tumor-derived systemic factors can influence LNCaP growth. Mice bearing LNCaP tumors with or without NE allografts were castrated 2 weeks after NE tumor inoculation, and changes in LNCaP tumor growth rate and gene expression were investigated. After castration, LNCaP tumor growth decreased in mice bearing LNCaP tumors alone, and this was accompanied by a loss of nuclear androgen receptor (AR) localization. In contrast, in castrated mice bearing both LNCaP and NE-10 tumors, LNCaP tumors continued to grow, had increased levels of nuclear AR, and secreted prostate-specific antigen. Therefore, in the absence of testicular androgens, NE secretions were sufficient to maintain LNCaP cell growth and androgen-regulated gene expression in vivo. Furthermore, in vitro experiments showed that NE secretions combined with low levels of androgens activated the AR, an effect that was blocked by the antiandrogen bicalutamide. Because an increase in AR level has been reported to be sufficient to account for hormone refractory prostate cancers, the NE cell population ability to increase AR level/activity can be another mechanism that allows prostate cancer to escape androgen ablation therapy.