The Differential Paracrine Role of the Endothelium in Prostate Cancer Cells.

The survival of patients with solid tumors, such as prostate cancer (PCa), has been limited and fleeting with anti-angiogenic therapies. It was previously thought that the mechanism by which the vasculature regulates tumor growth was driven by a passive movement of oxygen and nutrients to the tumor tissue. However, previous evidence suggests that endothelial cells have an alternative role in changing the behavior of tumor cells and contributing to cancer progression. Determining the impact of molecular signals/growth factors released by endothelial cells (ECs) on established PCa cell lines in vitro and in vivo could help to explain the mechanism by which ECs regulate tumor growth. Using cell-conditioned media collected from HUVEC (HUVEC-CM), our data show the stimulated proliferation of all the PCa cell lines tested. However, in more aggressive PCa cell lines, HUVEC-CM selectively promoted migration and invasion in vitro and in vivo. Using a PCa-cell-line-derived xenograft model co-injected with HUVEC or preincubated with HUVEC-CM, our results are consistent with the in vitro data, showing enhanced tumor growth, increased tumor microvasculature and promoted metastasis. Gene set enrichment analyses from RNA-Seq gene expression profiles showed that HUVEC-CM induced a differential effect on gene expression when comparing low versus highly aggressive PCa cell lines, demonstrating epigenetic and migratory pathway enrichments in highly aggressive PCa cells. In summary, paracrine stimulation by HUVEC increased PCa cell proliferation and tumor growth and selectively promoted migration and metastatic potential in more aggressive PCa cell lines.

D-Propranolol Impairs EGFR Trafficking and Destabilizes Mutant p53 Counteracting AKT Signaling and Tumor Malignancy.

Cancer therapy may be improved by the simultaneous interference of two or more oncogenic pathways contributing to tumor progression and aggressiveness, such as EGFR and p53. Tumor cells expressing gain-of-function (GOF) mutants of p53 (mutp53) are usually resistant to EGFR inhibitors and display invasive migration and AKT-mediated survival associated with enhanced EGFR recycling. D-Propranolol (D-Prop), the non-beta blocker enantiomer of propranolol, was previously shown to induce EGFR internalization through a PKA inhibitory pathway that blocks the recycling of the receptor. Here, we first show that D-Prop decreases the levels of EGFR at the surface of GOF mutp53 cells, relocating the receptor towards recycling endosomes, both in the absence of ligand and during stimulation with high concentrations of EGF or TGF-α. D-Prop also inactivates AKT signaling and reduces the invasive migration and viability of these mutp53 cells. Unexpectedly, mutp53 protein, which is stabilized by interaction with the chaperone HSP90 and mediates cell oncogenic addiction, becomes destabilized after D-Prop treatment. HSP90 phosphorylation by PKA and its interaction with mutp53 are decreased by D-Prop, releasing mutp53 towards proteasomal degradation. Furthermore, a single daily dose of D-Prop reproduces most of these effects in xenografts of aggressive gallbladder cancerous G-415 cells expressing GOF R282W mutp53, resulting in reduced tumor growth and extended mice survival. D-Prop then emerges as an old drug endowed with a novel therapeutic potential against EGFR- and mutp53-driven tumor traits that are common to a large variety of cancers.

The Inflammatory Profile of the Tumor Microenvironment, Orchestrated by Cyclooxygenase-2, Promotes Epithelial-Mesenchymal Transition.

The tumor microenvironment (TME) corresponds to a complex and dynamic interconnection between the extracellular matrix and malignant cells and their surrounding stroma composed of immune and mesenchymal cells. The TME has constant cellular communication through cytokines that sustain an inflammatory profile, which favors tumor progression, angiogenesis, cell invasion, and metastasis. Although the epithelial-mesenchymal transition (EMT) represents a relevant metastasis-initiating event that promotes an invasive phenotype in malignant epithelial cells, its relationship with the inflammatory profile of the TME is poorly understood. Previous evidence strongly suggests that cyclooxygenase-2 (COX-2) overexpression, a pro-inflammatory enzyme related to chronic unresolved inflammation, is associated with common EMT-signaling pathways. This review article summarizes how COX-2 overexpression, within the context of the TME, orchestrates the EMT process and promotes initial metastatic-related events.

Dietary Fructose Promotes Prostate Cancer Growth.

Clinical localization of primary tumors and sites of metastasis by PET is based on the enhanced cellular uptake of 2-deoxy-2-[18F]-fluoro-D-glucose (FDG). In prostate cancer, however, PET-FDG imaging has shown limited clinical applicability, suggesting that prostate cancer cells may utilize hexoses other than glucose, such as fructose, as the preferred energy source. Our previous studies suggested that prostate cancer cells overexpress fructose transporters, but not glucose transporters, compared with benign cells. Here, we focused on validating the functional expression of fructose transporters and determining whether fructose can modulate the biology of prostate cancer cells in vitro and in vivo. Fructose transporters, Glut5 and Glut9, were significantly upregulated in clinical specimens of prostate cancer when compared with their benign counterparts. Fructose levels in the serum of patients with prostate cancer were significantly higher than healthy subjects. Functional expression of fructose transporters was confirmed in prostate cancer cell lines. A detailed kinetic characterization indicated that Glut5 represents the main functional contributor in mediating fructose transport in prostate cancer cells. Fructose stimulated proliferation and invasion of prostate cancer cells in vitro. In addition, dietary fructose increased the growth of prostate cancer cell line-derived xenograft tumors and promoted prostate cancer cell proliferation in patient-derived xenografts. Gene set enrichment analysis confirmed that fructose stimulation enriched for proliferation-related pathways in prostate cancer cells. These results demonstrate that fructose promotes prostate cancer cell growth and aggressiveness in vitro and in vivo and may represent an alternative energy source for prostate cancer cells. SIGNIFICANCE: This study identifies increased expression of fructose transporters in prostate cancer and demonstrates a role for fructose as a key metabolic substrate supporting prostate cancer cells, revealing potential therapeutic targets and biomarkers.

Functional and genomic characterization of three novel cell lines derived from a metastatic gallbladder cancer tumor.

BACKGROUND: Gallbladder cancer (GBC) is the most common tumor of the biliary tract. The incidence of GBC shows a large geographic variability, being particularly frequent in Native American populations. In Chile, GBC represents the second cause of cancer-related death among women. We describe here the establishment of three novel cell lines derived from the ascitic fluid of a Chilean GBC patient, who presented 46% European, 36% Mapuche, 12% Aymara and 6% African ancestry. RESULTS: After immunocytochemical staining of the primary cell culture, we isolated and comprehensively characterized three independent clones (PUC-GBC1, PUC-GBC2 and PUC-GBC3) by short tandem repeat DNA profiling and RNA sequencing as well as karyotype, doubling time, chemosensitivity, in vitro migration capability and in vivo tumorigenicity assay. Primary culture cells showed high expression of CK7, CK19, CA 19-9, MUC1 and MUC16, and negative expression of mesothelial markers. The three isolated clones displayed an epithelial phenotype and an abnormal structure and number of chromosomes. RNA sequencing confirmed the increased expression of cytokeratin and mucin genes, and also of TP53 and ERBB2 with some differences among the three cells lines, and revealed a novel exonic mutation in NF1. The PUC-GBC3 clone was the most aggressive according to histopathological features and the tumorigenic capacity in NSG mice. CONCLUSIONS: The first cell lines established from a Chilean GBC patient represent a new model for studying GBC in patients of Native American descent.

Functional and genomic characterization of three novel cell lines derived from a metastatic gallbladder cancer tumor

BACKGROUND: Gallbladder cancer (GBC) is the most common tumor of the biliary tract. The incidence of GBC shows a large geographic variability, being particularly frequent in Native American populations. In Chile, GBC represents the second cause of cancer-related death among women. We describe here the establishment of three novel cell lines derived from the ascitic fluid of a Chilean GBC patient, who presented 46% European, 36% Mapuche, 12% Aymara and 6% African ancestry. RESULTS: After immunocytochemical staining of the primary cell culture, we isolated and comprehensively characterized three independent clones (PUC-GBC1, PUC-GBC2 and PUC-GBC3) by short tandem repeat DNA profiling and RNA sequencing as well as karyotype, doubling time, chemosensitivity, in vitro migration capability and in vivo tumorigenicity assay. Primary culture cells showed high expression of CK7, CK19, CA 19-9, MUC1 and MUC16, and negative expression of mesothelial markers. The three isolated clones displayed an epithelial phenotype and an abnormal structure and number of chromosomes. RNA sequencing confirmed the increased expression of cytokeratin and mucin genes, and also of TP53 and ERBB2 with some differences among the three cells lines, and revealed a novel exonic mutation in NF1. The PUC-GBC3 clone was the most aggressive according to histopathological features and the tumorigenic capacity in NSG mice. CONCLUSIONS: The first cell lines established from a Chilean GBC patient represent a new model for studying GBC in patients of Native American descent.

Estilo de vida y agresividad del cáncer de próstata / Lifestyle and aggressiveness of prostate cancer

El objetivo de este estudio fue establecer una asociación entre diversas variables demográficas y epidemiológicas con la agresividad del cáncer de próstata (CaP). Métodos: pacientes diagnosticados con CaP respondieron una encuesta que incluye el nivel de educación, los factores de riesgo cardiovascular (FRCV), los antecedentes familiares (HF) de CaP, consumo de alcohol, tabaquismo y otros. Se utilizó análisis univariado y multivariado (AMV) para establecer si los factores mencionados anteriormente afectan las variables asociadas con la agresividad del CaP, como la edad al momento del diagnóstico, el índice de Gleason, los márgenes positivos (MP) y las metástasis óseas (MO), entre otras. Resultados: se incluyeron ciento setenta y dos hombres en el análisis. Los pacientes con HF fueron diagnosticados a edades más tempranas que los pacientes sin HF (55,73 vs 66,45 años, p = 0,0001). Los pacientes que beben tienen un mayor número de MP que los pacientes que no (15 vs 4 pacientes, p = 0,04). El AMV mostró que los pacientes que consumen alcohol y los que fuman (activos o suspendidos) tuvieron un mayor riesgo de MP (OR = 4,45 y 4,1, IC 95 por ciento 1,16-17,07 y 1,14-14,72, respectivamente, ambos p <0,05). Los pacientes con mayor nivel de educación presentaron un mayor riesgo de CaP confinado (OR = 3,42, IC 95 por ciento 1,392-8,434, p = 0,007). Conclusiones: los pacientes que consumen alcohol, fuman y tienen un menor nivel de educación presentaron un mayor riesgo de desarrollar CaP agresivo. (AU)

The aim of this study was to establish an association between various demographic and epidemiological variables with aggressiveness of prostate cancer (PCa). Methods: Patients diagnosed with PCa, answered a survey that include level of education, cardiovascular risk factors (CVRF), family history (FH) of PCa, alcohol intake, smoke and others. Univariate and multivariate analysis (MVA) were used to establish whether the factors mentioned above affect variables associated with aggressiveness of PCa such as: age at diagnosis, Gleason score, positive margins (PM), and bone metastasis (BM). Results: One hundred and seventy two men were included in the analysis. Patients with FH had cancer diagnosed at younger ages (55.73 years to FH vs 66.45 years to no FH, p = 0.0001). Patients who drink had higher number of PM than patients who did not (15 vs 4 patients, p= 0.04). MVA showed that patients who consumed alcohol and patients who smoked (active or suspended) had an increased risk of PM (OR= 4.45 and 4.1, 95 percent CI 1.16-17.07 and 1.14-14.72, respectively, both p<0.05). Patients with higher level of education presented an increased risk of confined PCa (OR= 3.42, 95 percent CI 1.392-8.434, p= 0.007). Conclusions: Patients who consume alcohol, smoke and have lower level of education presented a higher risk of developing aggressive PCa. (AU)

Resistance of leukemia cells to cytarabine chemotherapy is mediated by bone marrow stroma, involves cell-surface equilibrative nucleoside transporter-1 removal and correlates with patient outcome.

The interaction between acute myeloid leukemia cells (AML) with the bone marrow stroma cells (BMSCs) determines a protective environment that favors tumor development and resistance to conventional chemotherapy. We showed that BMSCs secrete soluble factors that protect AML cells from Ara-C induced cytotoxicity. This leukemia chemoresistance is associated with a decrease in the equilibrative nucleoside transporter (ENT1) activity by inducing removal of ENT1 from the cell surface. Reduction of cell proliferation was also observed with activation of AKT and mTOR-dependent cell survival pathways, which may also contribute to the tumor chemoprotection. Analysis of primary BMSC cultures has demonstrated that AML patients with stroma capable to confer Ara-C resistance in vitro compared to AML patients without this stroma capacity were associated with a worse prognosis. The two year overall survival rate was 0% versus 80% respectively (p=0.0001). This is the first report of a chemoprotection mechanism based on the removal of a drug transporter from the cell surface and most importantly the first time that a stroma phenotype has correlated with prognostic outcome in cancer.

Androgens modulate male-derived endothelial cell homeostasis using androgen receptor-dependent and receptor-independent mechanisms.

BACKGROUND: Sex-related differences in the role of androgen have been reported in cardiovascular diseases and angiogenesis. Moreover, androgen receptor (AR) has been causally involved in the homeostasis of human prostate endothelial cells. However, levels of expression, functionality and biological role of AR in male- and female-derived human endothelial cells (ECs) remain poorly characterized. The objectives of this work were (1) to characterize the functional expression of AR in male- and female-derived human umbilical vein endothelial cell (HUVEC), and (2) to specifically analyze the biological effects of DHT, and the role of AR on these effects, in male-derived HUVECs (mHUVECs). RESULTS: Immunohistochemical analyses of tissue microarrays from benign human tissues confirmed expression of AR in ECs from several androgen-regulated and non-androgen-regulated human organs. Functional expression of AR was validated in vitro in male- and female-derived HUVECs using quantitative RT-PCR, immunoblotting and AR-mediated transcriptional activity assays. Our results indicated that functional expression of AR in male- and female-derived HUVECs was heterogeneous, but not sex dependent. In parallel, we analyzed in depth the biological effects of DHT, and the role of AR on these effects, on proliferation, survival and tube formation capacity in mHUVECs. Our results indicated that DHT did not affect mHUVEC survival; however, DHT stimulated mHUVEC proliferation and suppressed mHUVEC tube formation capacity. While the effect of DHT on proliferation was mediated through AR, the effect of DHT on tube formation did not depend on the presence of a functional AR, but rather depended on the ability of mHUVECs to further metabolize DHT. CONCLUSIONS: (1) Heterogeneous expression of AR in male- and female-derived HUVEC could define the presence of functionally different subpopulations of ECs that may be affected differentially by androgens, which could explain, at least in part, the pleiotropic effects of androgen on vascular biology, and (2) DHT, and metabolites of DHT, generally thought to represent progressively more hydrophilic products along the path to elimination, may have differential roles in modulating the biology of human ECs through AR-dependent and AR-independent mechanisms, respectively.

Selective targeting of bioengineered platelets to prostate cancer vasculature: new paradigm for therapeutic modalities.

Androgen deprivation therapy (ADT) provides palliation for most patients with advanced prostate cancer (CaP); however, greater than 80% subsequently fail ADT. ADT has been indicated to induce an acute but transient destabilization of the prostate vasculature in animal models and humans. Human re-hydrated lyophilized platelets (hRL-P) were investigated as a prototype for therapeutic agents designed to target selectively the tumour-associated vasculature in CaP. The ability of hRL-P to bind the perturbed endothelial cells was tested using thrombin- and ADP-activated human umbilical vein endothelial cells (HUVEC), as well as primary xenografts of human prostate tissue undergoing acute vascular involution in response to ADT. hRL-P adhered to activated HUVEC in a dose-responsive manner. Systemically administered hRL-P, and hRL-P loaded with super-paramagnetic iron oxide (SPIO) nanoparticles, selectively targeted the ADT-damaged human microvasculature in primary xenografts of human prostate tissue. This study demonstrated that hRL-P pre-loaded with chemo-therapeutics or nanoparticles could provide a new paradigm for therapeutic modalities to prevent the rebound/increase in prostate vasculature after ADT, inhibiting the transition to castration-recurrent growth.

Regulation of prostate stromal fibroblasts by the PIM1 protein kinase.

The PIM1 oncogene is over-expressed in human prostate cancer epithelial cells. Importantly, we observe that in human hyperplastic and cancerous prostate glands PIM1 is also markedly elevated in prostate fibroblasts, suggesting an important role for this kinase in epithelial/stromal crosstalk. The ability of PIM1 to regulate the biologic activity of stromal cells is demonstrated by the observation that expression of PIM1 kinase in human prostate fibroblasts increases the level and secretion of the extracellular matrix molecule, collagen 1A1 (COL1A1), the pro-inflammatory chemokine CCL5, and the platelet-derived growth factor receptors (PDGFR). PIM1 is found to regulate the transcription of CCL5. In co-cultivation assays where PIM1 over-expressing fibroblasts are grown with BPH1 prostate epithelial cells, PIM1 activity markedly enhances the ability of these fibroblasts to differentiate into myofibroblasts and express known markers of cancer-associated fibroblasts (CAFs). This differentiation can be reversed by the addition of small molecule PIM kinase inhibitors. Western blots demonstrate that PIM1 expression in prostate fibroblasts stimulates the phosphorylation of molecules that regulate 5'Cap driven protein translation, including 4EBP1 and eIF4B. Consistent with the hypothesis that the kinase controls translation of specific mRNAs in prostate fibroblasts, we demonstrate that PIM1 expression markedly increases the level of COL1A1 and PDGFRß mRNA bound to polysomes. Together these results point on PIM1 as a novel factor in regulation of the phenotype and differentiation of fibroblasts in prostate cancer by controlling both the transcription and translation of specific mRNAs.

Association of a single-nucleotide polymorphism from chromosome 17q12 with the aggressiveness of prostate cancer in a Hispanic population.

PURPOSE: To study the association between the polymorphisms, rs1859962 and rs4430796, from the chromosomes 17q24 and 17q12, respectively, with the risk of prostate cancer (PCa) and its clinical characteristics in a Hispanic (Chilean) population. METHODS: This study included 33 controls and 167 patients diagnosed with PCa. The polymorphisms, rs1859962 and rs4430796, were analyzed on blood specimens using quantitative PCR. The genetic analysis of the qPCR data was performed using the SNPStats program. A comparison between the clinical characteristics of the prostate cancers from the patients and the presence of the different polymorphism genotypes detected in blood specimens obtained from these patients was performed using the IBM SPSS v20.0 software. RESULTS: We observed no association of the SNPs and the risk of developing PCa (OR 0.84, 95 % CI 0.30-2.38, p = 1.0 to rs1859962 and OR 1.94, 95 % CI 0.57-6.52, p = 0.28 to rs4430796), both sporadic and hereditary. However, patients carrying the genotype G/G from the polymorphism rs4430796 had significantly higher PSA levels than patients carrying the other genotypes (15.05 ng/ml to G/G, 10 and 8.11 ng/ml to genotypes A/G y A/A, respectively, p = 0.01). Furthermore, patients with the genotype G/G of rs4430796 had higher tumor volume than other genotypes (9.45 cc to G/G and 5.22 cc to A/G + A/A, p = 0.04). CONCLUSION: The polymorphism rs4430796 of the chromosome 17q12 appears to be a biomarker for cancer aggressiveness, increased PSA and tumor volume of PCa.

Association of RNASEL and 8q24 variants with the presence and aggressiveness of hereditary and sporadic prostate cancer in a Hispanic population.

To study the association between the polymorphisms Arg462Gln and Asp541Glu from the RNASEL gene (1q25), and the polymorphisms rs620861, rs1447295, rs6983267, rs7837328 from the chromosome 8q24 with the risk of presenting prostate cancer (PCa) and its clinical characteristics in a Hispanic (Chilean) population. The study was performed on 21 control patients and 83 patients diagnosed with PCa. Polymorphisms were analysed from blood samples through real-time PCR by using TaqMan probes, and the genetic analysis was performed with the SNPStats program. Also, a comparison was performed between clinical characteristics of PCa and the presence of the different polymorphism genotypes by using the Minitab software. There was a significant association between the genotype G/G from the polymorphism rs6983267 with an overall increased risk of PCa, in patients both with or without family history of PCa (OR = 4.47, 95% CI = 1.05-18.94, P = 0.034 and OR = 3.57, 95% CI = 0.96-13.35, P = 0.037, respectively). Regarding clinical parameters, patients carrying the genotype C/C from the polymorphism Asp541Glu had significantly higher prostate-specific antigen (PSA) levels than patients carrying the other genotypes (P = 0.034). Moreover, patients with the genotype G/G of rs6983267 had higher PSA levels (P = 0.024). The polymorphism rs6983267 from region 3 of the chromosome 8q24 appears to be a prominent risk factor for PCa and a biomarker for cancer aggressiveness in the group of patients who presented higher levels of PSA at the time of diagnosis.

Role of androgen and vitamin D receptors in endothelial cells from benign and malignant human prostate.

Forty years ago, Judah Folkman (Folkman. N Engl J Med 285: 1182-1186, 1971) proposed that tumor growth might be controlled by limiting formation of new blood vessels (angiogenesis) needed to supply a growing tumor with oxygen and nutrients. To this end, numerous "antiangiogenic" agents have been developed and tested for therapeutic efficacy in cancer patients, including prostate cancer (CaP) patients, with limited success. Despite the lack of clinical efficacy of lead anti-angiogenic therapeutics in CaP patients, recent published evidence continues to support the idea that prostate tumor vasculature provides a reasonable target for development of new therapeutics. Particularly relevant to antiangiogenic therapies targeted to the prostate is the observation that specific hormones can affect the survival and vascular function of prostate endothelial cells within normal and malignant prostate tissues. Here, we review the evidence demonstrating that both androgen(s) and vitamin D significantly impact the growth and survival of endothelial cells residing within prostate cancer and that systemic changes in circulating androgen or vitamin D drastically affect blood flow and vascularity of prostate tissue. Furthermore, recent evidence will be discussed about the expression of the receptors for both androgen and vitamin D in prostate endothelial cells that argues for direct effects of these hormone-activated receptors on the biology of endothelial cells. Based on this literature, we propose that prostate tumor vasculature represents an unexplored target for modulation of tumor growth. A better understanding of androgen and vitamin D effects on prostate endothelial cells will support development of more effective angiogenesis-targeting therapeutics for CaP patients.

Dynamic expression of the sodium-vitamin C co-transporters, SVCT1 and SVCT2, during perinatal kidney development.

Isoform 1 of the sodium-vitamin C co-transporter (SVCT1) is expressed in the apical membrane of proximal tubule epithelial cells in adult human and mouse kidneys. This study is aimed at analyzing the expression and function of SVCTs during kidney development. RT-PCR and immunohistochemical analyses revealed that SVCT1 expression is increased progressively during postnatal kidney development. However, SVCT1 transcripts were barely detected, if not absent, in the embryonic kidney. Instead, the high-affinity transporter, isoform 2 (SVCT2), was strongly expressed in the developing kidney from E15; its expression decreased at postnatal stages. Immunohistochemical analyses showed a dynamic distribution of SVCT2 in epithelial cells during kidney development. In renal cortex tubular epithelial cells, intracellular distribution of SVCT2 was observed at E19 with distribution in the basolateral membrane at P1. In contrast, SVCT2 was localized to the apical and basolateral membranes between E17 and E19 in medullary kidney tubular cells but was distributed intracellularly at P1. In agreement with these findings, functional expression of SVCT2, but not SVCT1 was detected in human embryonic kidney-derived (HEK293) cells. In addition, kinetic analysis suggested that an ascorbate-dependent mechanism accounts for targeted SVCT2 expression in the developing kidney during medullary epithelial cell differentiation. However, during cortical tubular differentiation, SVCT1 was induced and localized to the apical membrane of tubular epithelial cells. SVCT2 showed a basolateral polarization only for the first days of postnatal life. These studies suggest that the uptake of vitamin C mediated by different SVCTs plays differential roles during the ontogeny of kidney tubular epithelial cells.

Altered corepressor SMRT expression and recruitment to target genes as a mechanism that change the response to androgens in prostate cancer progression.

Androgen receptor (AR) is required for the development and progression of prostate cancer (CaP) from androgen-dependence to androgen-resistance. Both corepressors and coactivators regulate AR-mediated transcriptional activity, and aberrant expression or activity due to mutation(s) contributes to changes in AR function in the progression to androgen resistance acquired during hormonal ablation therapies. Primary culture of epithelial cells from androgen-dependent CWR22 and androgen-resistant CWR22R xenograft tumors were used to evaluate the effect of androgens on AR function, and the association with coactivators (SRC-1 and TIF-2) and corepressors (SMRT and NCoR). Both androgen-dependent CWR22 and androgen-resistant CWR22R cells expressed functional AR as the receptor bind ligand with high affinity and increased trafficking to the nuclei in the presence of androgens. However, in the presence of androgens, AR-mediated transcriptional activity in androgen-sensitive CWR22 cells was limited to a 2-fold increase, as compared to a 6-fold increase in androgen-resistance CWR22R cells. In androgen-sensitive CWR22 cells, immunoblot, confocal microscopy, and chromatin immunoprecipitation assays indicated that the androgen bound AR transcriptional initiation complex in the PSA promoter contained corepressor SMRT, resulting in limited receptor transcriptional activity. In contrast, increased AR-mediated transcriptional activity in the CWR22R cells was consistent with decreased expression and recruitment of the corepressors SMRT/NCoR, as well as increased recruitment of the coactivator TIF-2 to the receptor complex. Similar changes in the response to androgens were observed in the LNCaP/C4-2 model of androgen resistance prostate cancer. Thus, altered recruitment and loss of corepressors SMRT/NCoR may provide a mechanism that changes the response of AR function to ligands and contributes to the progression of the advanced stages of human prostate cancer.

Bone marrow stromal cells modulate mouse ENT1 activity and protect leukemia cells from cytarabine induced apoptosis.

BACKGROUND: Despite a high response rate to chemotherapy, the majority of patients with acute myeloid leukemia (AML) are destined to relapse due to residual disease in the bone marrow (BM). The tumor microenvironment is increasingly being recognized as a critical factor in mediating cancer cell survival and drug resistance. In this study, we propose to identify mechanisms involved in the chemoprotection conferred by the BM stroma to leukemia cells. METHODS: Using a leukemia mouse model and a human leukemia cell line, we studied the interaction of leukemia cells with the BM microenvironment. We evaluated in vivo and in vitro leukemia cell chemoprotection to different cytotoxic agents mediated by the BM stroma. Leukemia cell apoptosis was assessed by flow cytometry and western blotting. The activity of the equilibrative nucleoside transporter 1 (ENT1), responsible for cytarabine cell incorporation, was investigated by measuring transport and intracellular accumulation of (3)H-adenosine. RESULTS: Leukemia cell mobilization from the bone marrow into peripheral blood in vivo using a CXCR4 inhibitor induced chemo-sensitization of leukemia cells to cytarabine, which translated into a prolonged survival advantage in our mouse leukemia model. In vitro, the BM stromal cells secreted a soluble factor that mediated significant chemoprotection to leukemia cells from cytarabine induced apoptosis. Furthermore, the BM stromal cell supernatant induced a 50% reduction of the ENT1 activity in leukemia cells, reducing the incorporation of cytarabine. No protection was observed when radiation or other cytotoxic agents such as etoposide, cisplatin and 5-fluorouracil were used. CONCLUSION: The BM stroma secretes a soluble factor that significantly protects leukemia cells from cytarabine-induced apoptosis and blocks ENT1 activity. Strategies that modify the chemo-protective effects mediated by the BM microenvironment may enhance the benefit of conventional chemotherapy for patients with AML.

Primary xenografts of human prostate tissue as a model to study angiogenesis induced by reactive stroma.

Characterization of the mechanism(s) of androgen-driven human angiogenesis could have significant implications for modeling new forms of anti-angiogenic therapies for CaP and for developing targeted adjuvant therapies to improve efficacy of androgen-deprivation therapy. However, models of angiogenesis by human endothelial cells localized within an intact human prostate tissue architecture are until now extremely limited. This report characterizes the burst of angiogenesis by endogenous human blood vessels in primary xenografts of fresh surgical specimens of benign prostate or prostate cancer (CaP) tissue that occurs between Days 6-14 after transplantation into SCID mice pre-implanted with testosterone pellets. The wave of human angiogenesis was preceded by androgen-mediated up-regulation of VEGF-A expression in the stromal compartment. The neo-vessel network anastomosed to the host mouse vascular system between Days 6-10 post-transplantation, the angiogenic response ceased by Day 15, and by Day 30 the vasculature had matured and stabilized, as indicated by a lack of leakage of serum components into the interstitial tissue space and by association of nascent endothelial cells with mural cells/pericytes. The angiogenic wave was concurrent with the appearance of a reactive stroma phenotype, as determined by staining for α-SMA, Vimentin, Tenascin, Calponin, Desmin and Masson's trichrome, but the reactive stroma phenotype appeared to be largely independent of androgen availability. Transplantation-induced angiogenesis by endogenous human endothelial cells present in primary xenografts of benign and malignant human prostate tissue was preceded by induction of androgen-driven expression of VEGF by the prostate stroma, and was concurrent with and the appearance of a reactive stroma phenotype. Androgen-modulated expression of VEGF-A appeared to be a causal regulator of angiogenesis, and possibly of stromal activation, in human prostate xenografts.

Biochemical characterization of nuclear receptors for vitamin D3 and glucocorticoids in prostate stroma cell microenvironment.

The disruption of stromal cell signals in prostate tissue microenvironment influences the development of prostate cancer to androgen independence. 1α,25-Dihydroxyvitamin D(3) (1,25D(3)) and glucocorticoids, either alone or in combination, have been investigated as alternatives for the treatment of advanced prostate cancers that fails androgen therapies. The effects of glucocorticoids are mediated by the intracellular glucocorticoid receptor (GR). Similarly, the effect of 1,25D(3) is mediated by the 1,25D(3) nuclear receptor (VDR). In this study, fibroblasts from benign- (BAS) and carcinoma-associated stroma (CAS) were isolated from human prostates to characterize VDR and GR function as transcription factors in prostate stroma. The VDR-mediated transcriptional activity assessed using the CYP24-luciferase reporter was limited to 3-fold induction by 1,25D(3) in 9 out of 13 CAS (70%), as compared to >10-fold induction in the BAS clinical sample pair. Expression of His-tagged VDR (Ad-his-VDR) failed to recover the low transcriptional activity of the luciferase reporter in 7 out of 9 CAS. Interestingly, expression of Ad-his-VDR successfully recovered receptor-mediated induction in 2 out of the 9 CAS analyzed, suggesting that changes in the receptor protein itself was responsible for decreased response and resistance to 1,25D(3) action. Conversely, VDR-mediated transcriptional activity was more efficient in 4 out of 13 CAS (30%), as compared to the BAS sample pair. Consistent with the reduced response to 1,25D(3) observed in CAS, chromatin immunoprecipitation (ChIP) assays indicated decreased recruitment of coactivators SRC-1/CBP, without major changes in the recruitment of VDR to the CYP24 promoter. In addition, we observed that GR-mediated transcriptional activity was also altered in CAS, as compared to BAS. Disruption of coactivators SRC-1/CBP recruitment may promote hormone resistance in CaP, and highlights the relevance of molecular diagnosis and drug design in tumor cell microenvironment.

Androgen deprivation induces rapid involution and recovery of human prostate vasculature.

The response of the prostate tissue microenvironment to androgen deprivation (AD) represents a critical component in the treatment of benign prostatic hyperplasia and prostate cancer (CaP). Primary xenografts of human benign and CaP tissue transplanted to immunocompromized SCID mice were used to characterize the response of the prostate vasculature during the initial 14 days of AD. Microvessel density and vascular lumen diameter in the prostate xenografts decreased rapidly after AD, reached a nadir on days 2-4, and recovered between days 4 and 14. The number of apoptotic endothelial cells peaked on day 2 after AD and decreased to precastration levels over days 4-7. Leakage of vascular contents in the interstitial space was apparent between days 1 and 3 after AD; however, the vascular permeability barrier reestablished between days 7 and 14. Expression of vascular endothelial growth factor (VEGF)-A, VEGF receptor-2, and basic fibroblast growth factor protein increased in endothelial cells between days 2 and 4 after AD, which preceded vascular recovery and appeared to be a direct and specific response of the endothelial cells to AD. Lack of comparable upregulation of these genes in primary cultures of human prostate endothelial cells in response to AD suggests a role for paracrine signaling mediated through stromal or epithelial cells. VEGF-A expression by prostate endothelial cells appears to represent a key facilitator of the vascular rebound in human prostate tissue induced by removal of circulating testicular androgens.