Prostate Cancer Epithelial Cells Research Articles

The Transcription Factor ERG Regulates Super-Enhancers Associated With an Endothelial-Specific Gene Expression Program.

The ETS (E-26 transformation-specific) transcription factor ERG (ETS-related gene) is essential for endothelial homeostasis, driving expression of lineage genes and repressing proinflammatory genes. Loss of ERG expression is associated with diseases including atherosclerosis. ERG's homeostatic function is lineage-specific, because aberrant ERG expression in cancer is oncogenic. The molecular basis for ERG lineage-specific activity is unknown. Transcriptional regulation of lineage specificity is linked to enhancer clusters (super-enhancers). To investigate whether ERG regulates endothelial-specific gene expression via super-enhancers. Chromatin immunoprecipitation with high-throughput sequencing in human umbilical vein endothelial cells showed that ERG binds 93% of super-enhancers ranked according to H3K27ac, a mark of active chromatin. These were associated with endothelial genes such as DLL4 (Delta-like protein 4), CLDN5 (claudin-5), VWF (von Willebrand factor), and CDH5 (VE-cadherin). Comparison between human umbilical vein endothelial cell and prostate cancer TMPRSS2 (transmembrane protease, serine-2):ERG fusion-positive human prostate epithelial cancer cell line (VCaP) cells revealed distinctive lineage-specific transcriptome and super-enhancer profiles. At a subset of endothelial super-enhancers (including DLL4 and CLDN5), loss of ERG results in significant reduction in gene expression which correlates with decreased enrichment of H3K27ac and MED (Mediator complex subunit)-1, and reduced recruitment of acetyltransferase p300. At these super-enhancers, co-occupancy of GATA2 (GATA-binding protein 2) and AP-1 (activator protein 1) is significantly lower compared with super-enhancers that remained constant following ERG inhibition. These data suggest distinct mechanisms of super-enhancer regulation in endothelial cells and highlight the unique role of ERG in controlling a core subset of super-enhancers. Most disease-associated single nucleotide polymorphisms from genome-wide association studies lie within noncoding regions and perturb transcription factor recognition sequences in relevant cell types. Analysis of genome-wide association studies data shows significant enrichment of risk variants for cardiovascular disease and other diseases, at ERG endothelial enhancers and super-enhancers. The transcription factor ERG promotes endothelial homeostasis via regulation of lineage-specific enhancers and super-enhancers. Enrichment of cardiovascular disease-associated single nucleotide polymorphisms at ERG super-enhancers suggests that ERG-dependent transcription modulates disease risk.

The role of heterogeneous environment and docetaxel gradient in the emergence of polyploid, mesenchymal and resistant prostate cancer cells.

The ability of a population of PC3 prostate epithelial cancer cells to become resistant to docetaxel therapy and progress to a mesenchymal state remains a fundamental problem. The progression towards resistance is difficult to directly study in heterogeneous ecological environments such as tumors. In this work, we use a micro-fabricated "evolution accelerator" environment to create a complex heterogeneous yet controllable in-vitro environment with a spatially-varying drug concentration. With such a structure we observe the rapid emergence of a surprisingly large number of polyploid giant cancer cells (PGCCs) in regions of very high drug concentration, which does not occur in conventional cell culture of uniform concentration. This emergence of PGCCs in a high drug environment is due to migration of diploid epithelial cells from regions of low drug concentration, where they proliferate, to regions of high drug concentration, where they rapidly convert to PGCCs. Such a mechanism can only occur in spatially-varying rather than homogeneous environments. Further, PGCCs exhibit increased expression of the mesenchymal marker ZEB1 in the same high-drug regions where they are formed, suggesting the possible induction of an epithelial to mesenchymal transition (EMT) in these cells. This is consistent with prior work suggesting the PGCC cells are mediators of resistance in response to chemotherapeutic stress. Taken together, this work shows the key role of spatial heterogeneity and the migration of proliferative diploid cells to form PGCCs as a survival strategy for the cancer population, with implications for new therapies.

A Review of Prostate-Specific Membrane Antigen (PSMA) Positron Emission Tomography (PET) in Renal Cell Carcinoma (RCC).

Metastatic renal cell carcinoma (mRCC) is a disease that portends poor prognosis despite an increasing number of novel systemic treatment options including new targeted therapies and immunotherapy. Ablative intervention directed at oligometastatic RCC has demonstrated survival benefit. Consequently, developing techniques for improved staging of mRCC on contemporary imaging modalities including X-ray computed tomography (CT), magnetic resonance imaging (MRI) and/or bone scan (BS) is a clinical priority. This is relevant for metastatic deposits too small to characterize or lymph nodes within physiological normality. Prostate-specific membrane antigen (PSMA) is a type II transmembrane glycoprotein highly expressed on prostate cancer epithelial cells. Recently, small molecules targeting the PSMA receptor, linked to radioactive isotopes have been developed for use with positron emission tomography (PET). Despite its nomenclature, PSMA has also been found to be expressed in the neovasculature of non-prostate cancers such as renal cell carcinoma (RCC) and hence PSMA PET/CT imaging has been proposed as an alternative staging modality. Preliminary small studies involving the use of PSMA PET/CT imaging in mRCC have been encouraging with evidence of improved staging sensitivity which has directly led to change in management in some cases. Given these early encouraging reports, we performed a comprehensive narrative review on the available evidence, including the scientific basis for PSMA expression in RCC, the role of PSMA PET/CT imaging with potential clinical implications in mRCC, its limitations and future opportunities.

3D Co-culture System of Mouse Prostatic Wild-type Fibroblasts withHuman Prostate Cancer Epithelial Cells.

Heterogeneous prostatic carcinoma-associated fibroblasts (CAF) contribute to tumor progression. This was established using transgenic mouse models. Paracrine interactions between fibroblasts and epithelial cells were further interrogated using isolated 2D cell culture systems, but 3D culture systems currently being developed can better mimic reciprocal interactions potentially found in the native tissue. To understand paracrine and juxtacrine signaling among fibroblasts and epithelia, 3D co-cultures with species differences allows for further subsequent analysis of the cultures. The use of mouse and human cells, for example, in one system allows for species-specific FACS or quantitative PCR analysis. This protocol describes the use of a 3D Co-culture System of Mouse Prostatic Wild-type Fibroblasts with Human Prostate Cancer Epithelial Cells.

ITRAQ-based Comparative Serum Proteomic Analysis of Prostate Cancer Patients with or without Bone Metastasis.

Background: Once prostate cancer developed bone metastasis, the quality of life and prognosis of patients are seriously affected as no effective treatment is currently available. It is necessary to explore the mechanism of bone metastasis and new therapeutic targets.Purpose: To find out the differentially expressed serum proteins in prostate cancer patients with bone metastasis and analyze the expression of key proteins in prostate cancer tissues, serum and prostate cancer cell lines. So as to provide a basis for revealing the mechanism of bone metastasis and designing new therapeutic targets.Methods: iTRAQ-based proteomics method was used to compare serum differential proteins in prostate cancer patients with and without bone metastasis. Three key proteins (CD59, haptoglobin and tetranectin) which had significant fold changes were selected to validate the results of mass spectrometry. Immunohistochemistry and ELISA were applied to tissues and serum samples from prostate cancer patients, respectively, for validation. Finally, western blot, flow cytometry, and immunocytochemistry were used to analyze the expression of the three differentially expressed proteins in the prostate cancer cell lines PC3, LNCap, and Du145.Results: Thirty-two differentially expressed proteins related to bone metastasis of prostate cancer were identified, of which 11 were up-regulated and 21 were down-regulated. CD59 and haptoglobin were up-regulated in prostate cancer with bone metastasis while tetranectin was down-regulated. Tetranectin showed differential expression in epithelial and stromal cells of prostate cancer and hyperplasia tissues.The expression of CD59 was highest in PC3 and lowest in LNCap, while the expression of haptoglobin and tetranectin was the highest in DU145 and lowest in PC3.Conclusion: Mass spectrometry analysis showed that there were more differentially expressed proteins in the serum of patients with bone metastasis than those without metastasis. It has been verified that CD59, haptoglobin and tetranectin are prostate cancer bone metastasis related proteins.

African-American Prostate Normal and Cancer Cells for Health Disparities Research.

Prostate cancer is the most frequently diagnosed solid malignancy in men. Epidemiological studies have shown African-American men to be at higher risk for developing prostate cancer and experience higher death as compared to other ethnic groups. Establishment of prostate cancer cell lines paired with normal cells derived from the same patient is a fundamental breakthrough in cell culture technology and provides a resource to improve our understanding of cancer development and pertinent molecular events. Previous studies have demonstrated that conditional reprogramming (CR) allows the establishment and propagation of patient-derived normal and tumor epithelial cell cultures from a variety of tissue types. Here, we report a new AA prostate cell model, paired normal and cancer epithelial cells from the same patient. "Tumor" cell culture AA-103A was derived from malignant prostate tissues, and "normal" cell culture AA-103B was derived from non-malignant prostate tissues from the prostatectomy specimen of an African-American male. These paired cell cultures have been propagated under CRC conditions to permit direct comparison of the molecular and genetic profiles of the normal epithelium and adenocarcinoma cells for comparison of biomarkers, enabling patient-specific pathological analysis, and molecular and cellular characterization. STR confirmed human origin albeit no karyotypic abnormalities in the two cell lines. Further quantitative PCR analyses demonstrated characteristic markers, including the high level of basal cell marker, the keratin 5 (KRT5) in normal cells and of luminal marker, the androgen receptor (AR) as well as the programmed death-ligand 1 (PD-L1) in tumor cells. Although 3-D sphere formation was observed, the AA-103A of tumor cells did not generate tumors in vivo. We report these paired primary epithelial cultures under CRC growth as a potentially useful tool for studies to understand molecular mechanisms underlying health disparities in prostate cancer.

Purification and Functional Characterization of the C-Terminal Domain of the β-Actin-Binding Protein AIM1 In Vitro.

The protein absent in melanoma 1 (AIM1) is a member of the βγ-crystal lens superfamily that is associated with the development of multiple cancers. The binding of AIM1 to β-actin affects the migration and invasion of prostate cancer epithelial cells. The C-terminus of AIM1 is required for the β-actin interaction. However, the characteristics of AIM1 in vitro and the interaction mode between AIM1 and β-actin remain unknown. We describe novel methods to prepare pure recombinant AIM1 and identify possible binding modes between AIM1 and β-actin; we also obtain the crystal of the first two βγ-crystallin domains of AIM1 (g1g2) for future structural biology research. We first express and purify AIM1 after cloning the sequence into a modified pET-28a_psp expression vector. Next, we define the minimum unit formed by the βγ-crystallin domain repeats that bound to β-actin and perform its physiological function. Finally, we made the structural model of the AIM1 g1g2 that can be used to guide future biomedical investigations and prostate cancer research.

128 - RelB-BLNK axis: a novel determinant of cell fate

Prostate cancer (PCa), the second leading cause of male cancer deaths in the United States, is most frequently treated with radiation therapy (RT). We screened 786 FDA-approved compounds to identify drugs that can radiosensitize PCa cells while radioprotecting non-cancer prostate epithelial cells and identified Betamethasone (BET) as a lead compound with these properties. BET increases ROS levels in PCa cells (high oxidized redox state) but not in normal prostate epithelial cells (low oxidized redox state). Remarkably, BET has an opposite effect on RelB, a non-canonical redox-sensitive NF-κB pathway. BET suppresses the expression of RelB and RelB target genes including MnSOD and survivin in PCa cells, while it induces expression of RelB and MnSOD in non-cancer cells. The differential effects of BET are, in part, attributable to the intrinsic differences in the cellular redox states of normal versus PCa cells. Intriguingly, RNA seq and ChIP assays identify B-cell linker protein (BLNK) as a novel RelB transcription regulator that is differentially regulated by BET in normal and cancer cells. Proximity-ligation assays and knockdown of BLNK confirm that RelB-BLNK possibly interact in the nuclear compartment. BET/RT treatments that target disruptions of RelB and BLNK expressions reverse the survival of PCa cells but not of normal prostate epithelial cells. Our study is the first to establish that the RelB-BLNK mediated NF-κB non-canonical pathway is a novel mechanism to regulate the differential responses to radiation therapy of normal prostate epithelial and prostate cancer cells. The results of this study fill in a critical gap in scientific knowledge of how the differences in redox conditions of normal and tumor cells trigger differential responses to pro-oxidants. They will facilitate the translation of therapeutic approaches that use prooxidants to push cancer cells into oxidative stress overload but stimulate adaptive responses in normal cells to selectively enhance RT efficacy.

Bitter taste receptors are expressed in human epithelial ovarian and prostate cancers cells and noscapine stimulation impacts cell survival.

Bitter taste receptors (Tas2Rs) are a subfamily of G-protein coupled receptors expressed not only in the oral cavity but also in several extra-oral tissues and disease states. Several natural bitter compounds from plants, such as bitter melon extract and noscapine, have displayed anti-cancer effects against various cancer types. In this study, we examined the prevalence of Tas2R subtype expression in several epithelial ovarian or prostate cancer cell lines, and the functionality of Tas2R14 was determined. qPCR analysis of five TAS2Rs demonstrated that mRNA expression often varies greatly in cancer cells in comparison to normal tissue. Using receptor-specific siRNAs, we also demonstrated that noscapine stimulation of ovarian cancer cells increased apoptosis in ovarian cancer cells in a receptor-dependent, but ROS-independent manner. This study furthers our understanding of the function of Tas2Rs in ovarian cancer by demonstrating that their activation has an impact on cell survival.

Expression of LMO2 in Prostate Carcinoma and Adjacent Prostatic Parenchyma.

LMO2 (LIM domain only) is a member of transcription factor family of proteins characterized by their cysteine-rich, zinc-binding LIM domains. Its expression in prostate cancer cells, as well as in adjacent stroma, is described in a study in a cohort of 83 patients treated with radical prostatectomy for clinically localized prostate adenocarcinoma. Authors found that LMO2 overexpression in prostate cancer was strongly associated with features indicative of worse prognosis (higher preoperative PSA, higher Gleason score, positive surgical margins, and extraprostatic extension of disease). Expression of LMO2 was also associated with biochemical disease progression. We analysed immunohistochemical expression of LMO2 in prostate cancer epithelial and stromal cells, as well as in adjacent parenchyma. Significant negative correlation between glandular expression of LMO2 in carcinoma and stromal expression in BPH (ρ = -0.238, P = 0.033) was found, but also be-tween stromal expression in carcinomas and glandular expression in BPH (ρ = -0.255, P = 0.021). Positive correlation was found between stromal expression in BPH and stromal expression in carci-nomas (ρ = 0.306, P = 0.005). Study results support the potential role of LMO2 in prostatic carcino-genesis and cancer progression.

3D polyelectrolyte scaffolds to mimic exocrine glands: a step towards a prostate-on-chip platform

Abstract We report our approach to creating a microfluidic chip (namely UroLOC) that mimics the acinar/tubular structure and the luminal microenvironment of exocrine glands. The chip utilises a nanostructured membrane that is designed to provide a 3-dimensional supporting scaffold for the growth of exocrine acinus epithelial cells. The nanostructured membrane was produced using layer-by-layer assembly of polyelectrolytes, and formed into 3-dimensional hemispherical cavities and “finger-like” structures in order to mimic the natural architecture of acini found in exocrine glands. We utilised normal (PNT2) and cancerous (PC3, LNCaP) prostate epithelial cells to demonstrate the proof-of-concept of using MALDI (Matrix Assisted Laser Desorption Ionisation) profiling of secretions collected after 48 hours of cell growth, with no concentration or purification steps and without any a priori on the knowledge of targeted proteins. This MALDI profiling analysis of the crude supernatants from 3 different cell lines (PNT2, PC3 and LNCaP) demonstrated the capacity of the MALDI profiling approach to discriminate between the different secretome signatures. The UroLOC concept and secretome profiling that we describe opens new opportunities in terms of liquid-biopsy based diagnosis, particularly for the early stages of carcinogenesis.

3D Shape Modeling for Cell Nuclear Morphological Analysis and Classification

Quantitative analysis of morphological changes in a cell nucleus is important for the understanding of nuclear architecture and its relationship with pathological conditions such as cancer. However, dimensionality of imaging data, together with a great variability of nuclear shapes, presents challenges for 3D morphological analysis. Thus, there is a compelling need for robust 3D nuclear morphometric techniques to carry out population-wide analysis. We propose a new approach that combines modeling, analysis, and interpretation of morphometric characteristics of cell nuclei and nucleoli in 3D. We used robust surface reconstruction that allows accurate approximation of 3D object boundary. Then, we computed geometric morphological measures characterizing the form of cell nuclei and nucleoli. Using these features, we compared over 450 nuclei with about 1,000 nucleoli of epithelial and mesenchymal prostate cancer cells, as well as 1,000 nuclei with over 2,000 nucleoli from serum-starved and proliferating fibroblast cells. Classification of sets of 9 and 15 cells achieved accuracy of 95.4% and 98%, respectively, for prostate cancer cells, and 95% and 98% for fibroblast cells. To our knowledge, this is the first attempt to combine these methods for 3D nuclear shape modeling and morphometry into a highly parallel pipeline workflow for morphometric analysis of thousands of nuclei and nucleoli in 3D.

Abstract A063: Preclinical evaluation of an arginine vasopressin receptor 1A (AVPR1A) antagonist in castration-resistant prostate cancer

Abstract Background: Advanced prostate cancer is treated by androgen deprivation, but despite initial responses most tumors inevitably recur. The recurrent disease is termed castration-resistant prostate cancer (CRPC) and is characterized by active androgen receptor (AR) signaling despite decreased androgen levels. AR activity in CRPC occurs through a variety of mechanisms including upregulation of AR coactivators, such as Vav3, and expression of constitutively active AR variants, which lack the ligand binding domain and are linked to poor prognosis. We previously demonstrated that PC cells are growth inhibited by depletion of either Vav3 or the clinically prevalent AR variant, AR-V7 (1). Gene expression profiling in CRPC cells revealed that arginine vasopressin receptor 1a (AVPR1a) was significantly reduced by depletion of either Vav3 or AR-V7. AVPR1a is a G protein-coupled receptor that has not been previously linked to PC. Analysis of publicly available datasets showed that the AVPR1a gene exhibits a significant increase in copy number in human CRPC specimens and AVPR1a mRNA is upregulated in aggressive PC (2-4). Methods: To explore the role of AVPR1a in PC cells, we selectively depleted AVPR1a in a panel of prostate cancer and prostate epithelial cell lines and examined cell survival and proliferation in culture and soft agar, as well as cell migration and invasion. Conversely, we overexpressed AVPR1a in an androgen-dependent PC cell line, LNCaP, and examined growth in vitro and in vivo in androgen-depleted conditions. We investigated AVPR1a antagonism in two distinct in vivo models of CRPC progression and growth using an effective and safe-in-humans AVPR1a antagonist, relcovaptan. Results: We showed that depletion of AVPR1a greatly inhibited the proliferation of multiple CRPC cell lines but had less to no effect on androgen-dependent PC cells or nontumorigenic prostate epithelial cells. Depletion of AVPR1a also decreased CRPC cell anchorage-independent growth. Conversely, overexpression of AVPR1a in androgen-dependent LNCaP cells conferred castration resistance in vitro and in vivo. Similar to depletion of AVPR1a, treatment of PC cells with the AVPR1a antagonist relcovaptan decreased CRPC anchorage-independent growth. AVPR1a depletion or antagonism was also effective at decreasing CRPC metastatic properties: migration and invasion. In a preclinical xenograft model of progression to castration resistance, we found that relcovaptan halted tumor growth and stabilized circulating levels of a clinical tumor progression marker, prostate specific antigen (PSA). We evaluated relcovaptan efficacy on end-stage metastatic CRPC growth. C4-2B CRPC cells were injected into the tibias of castrated mice to model tumor growth and bone remodeling. Relcovaptan significantly inhibited tumor growth and prevented cancer cell-induced bone remodeling. Conclusion: AVPR1a is a promising new therapeutic target against CRPC. We propose that AVPR1a antagonists may be repurposed for PC therapy.

Abstract B097: NRF2 antioxidant signaling mediates the ability of PIM protein kinases to induce resistance of prostate cancer to PI3K inhibitor therapy

Abstract Aberrant activation of the PI3K pathway leading to increases in AKT activity is seen in many prostate cancer patients refractory to androgen blockade therapy. A number of small-molecule inhibitors of PI3K, including BKM120 (Novartis), have entered clinical trials. As with PI3K-AKT activation, highly expressed Pim kinase has been shown to drive the growth of prostate cancer. Recent studies have demonstrated that Pim kinase plays a critical role in promoting resistance of breast cancer to PI3K inhibition. Similarly, when the Pim-1 protein kinase is overexpressed, prostate cancer cell lines become resistant to PI3K inhibitor. In both tissue culture and animal models, the combination of a Pim inhibitor, PIM447, and BKM120 acted synergistically in blocking the survival and growth of prostate cancer cells. We therefore investigated the biochemical mechanism by which Pim-1 mediates cell resistance to the PI3K inhibitor. Previously, we have demonstrated that knockout of Pim kinases led to accumulation of reactive oxygen species (ROS) accompanied by a lower level of expression of ROS scavengers. To explore whether Pim-1 overexpression reverses this phenotype in prostate epithelial cancer, using lentiviral transduction, we expressed Pim-1 in mouse prostate epithelial cancer cells derived from spontaneous prostate adenocarcinomas that arose in a mouse model in which Tp53 and Rb1 are deleted in prostate epithelium. Pim-1 overexpression promoted anchorage-independent growth and formation of mouse prostate epithelial tumors in vivo. Gene expression arrays demonstrated that Pim-1 induced an Nrf2 antioxidant response element gene signature, including increases in Heme oxygenase-1 (Hmox1), NAD(P)H quinone oxidoreductase 1 (Nqo1), and glutathione S-transferase kappa1 (Gstk1) genes. Similarly, overexpression of Pim-1 in human prostate cancer LNCaP cells results in increased expression of Nrf2 and Hmox1 protein. As expected, depletion of Nrf2 with shRNA prevented Pim-1-mediated increases in Hmox1 expression, indicating that Nrf2 activation is required for the Pim-1 induction of ROS scavengers. In line with this result, in Pim-1 overexpressing cell lines knockdown of either Hmox1 or Nrf2 restored sensitivity to BKM120 treatment. A vital role of ROS scavengers in mediating the resistance to BKM120 was further confirmed by the treatment of cells and xenografted tumors with buthionine sulfoximine (inhibitor of glutathione synthesis), which resensitized the Pim-1 overexpressing cells to BKM120 via ROS induction. Our data further demonstrated that Pim-1 overexpression decreases the ubiquitination of Nrf2 protein, thus preventing its degradation, and increasing its half-life. Conversely, inhibition of Pim kinase promoted Nrf2 ubiquitination and proteasomal degradation. Taken together, our results show that the increased expression of Pim kinases in prostate cancer blocks therapeutic efficacy of PI3K inhibitor by inducing Nrf2 and antioxidant scavengers that inhibit the induction of ROS. Citation Format: Jin H. Song, Libia A. Leuvano, Neha Singh, Andrew S. Kraft. NRF2 antioxidant signaling mediates the ability of PIM protein kinases to induce resistance of prostate cancer to PI3K inhibitor therapy [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B097.

Abstract B086: Monocytes-produced Chitinase-3-like 1 is a driver of metastatic behavior in advanced prostate cancer patients

Abstract Purpose: The identification, functional status, distribution, and interactions of immune cells in PCa patients have yet to be fully characterized. Understanding these details could provide key insights as to how promising current and developing immunotherapies might be directed toward PCa. Recruited myeloid cells promote cancer initiation, malignant progression, metastasis, and resistance to therapy in the tumor niche. In the present study, we tested the hypothesis that circulating blood monocytes from advanced prostate cancer (PCa) patients exhibit a tumor-promoting phenotype and directly influence the tumor microenvironment in response to tumor-derived signals. Experimental Design: Monocytes from metastatic (PCa-M) and nonmetastatic (PCa-N) patient blood samples were isolated, cultured, and conditioned media (CM) was obtained. To evaluate the role of monocytes in metastatic behavior of PCa cells, in vitro invasion (via Matrigel) and motility assays (Incucyte Live Cell Analysis System) were performed using monocyte-CM. To identify the candidate PCa-M monocyte-secreted protein(s) that might be implicated in tumor progression, we analyzed the CM using Proteome Profiler Analysis (R&D). Data were validated using ELISA and qPCR. Results: Herein, we report that CM from circulating monocytes in patients with PCa/mCRPC (metastatic castration-resistant PCa) increased motility and invasion of epithelial PCa cells. Proteome Profiler Analysis revealed that monocyte-derived CM from metastatic PCa/mCRPC patients presented high levels of YKL-40 (Chitinase-3-like 1; CHI3L1) when compared with PCa-N patients and healthy controls (HC), classified as individuals with no known history of cancer. The only described receptor for YKL-40, interleukin-13 receptor α2 (IL-13Rα2), was significantly upregulated in ARCaPm cells; accordingly, we observed that the activation of IL-13Rα2 from PCa-M CM increased the invasiveness of ARCaPm. In addition, siRNA directed against this receptor in ARCaPm cells caused a significant reduction in the invasiveness of these cells in the presence of CM from PCa-M patients. Conclusions: We show that circulating monocytes from metastatic PCa/mCRPC patients exert a tumor-promoting role via the secretion of YKL-40, and YKL-40 demands further exploration as a possible therapeutic target in advanced PCa. Citation Format: Karen A. Cavassani, Rebecca Meza, David Habiel, Jie-Fu Chen, Alexander Montes, Manisha Tripathi, Gislaine Martins, Timothy R. Crother, Cory M. Hogaboam, Sungyong You, Neil Bhowmick, Edwin Posadas. Monocytes-produced Chitinase-3-like 1 is a driver of metastatic behavior in advanced prostate cancer patients [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B086.

Parallel study on protein O-GlcNAcylation in prostate cancer cell with a sensitive microarray biochip

Although a variety of approaches have been developed to analyze protein O-GlcNAcylation, efficient investigations on O-GlcNAcylation of proteins of interest in high-throughput manner are still in high demand to further explore its functionality. In this work, we first develop a powerful microarray platform for a sensitive, specific and high-throughput analysis of protein O-GlcNAcylation. The developed array biochip is then utilized to parallelly analyze the O-GlcNAcylation of three oncogenic transcription factors C-Myc, NF-κB and p53 in normal prostate epithelial cell (RWPE-1) and prostate cancer cell line (PC-3). The levels of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) are also monitored by the microarray platform. The experimental results show that the overall O-GlcNAcylation and OGT expression level are obviously elevated in PC-3 as compared to RWPE-1. The protein expression-normalized O-GlcNAcylation of C-Myc and NF-κB in PC-3 is significantly higher than that in RWPE-1, while opposite result is observed from p53. In addition, the biological behaviors including proliferation and migration of PC-3 cells are also studied when OGA inhibitor Thiamet G is applied to elevate the total O-GlcNAcylation level.

PO-183 Identification of differentially hypomethylated genes associated to metastasis behaviour in colorectal cancer

IntroductionThe transcription factor PRH/HHEX (Proline-Rich Homeodomain/Haematopoietically Expressed Homeobox) controls cell proliferation, cell differentiation and cell migration/invasion in a diverse range of cell types. Our recent work showed that PRH expression is down-regulated in breast and prostate cancer cells and that this results in increased cell proliferation and cancer cell invasion. We also showed that PRH inhibits breast and prostate cancer cell migration and invasion in part at least by activating the transcription of Endoglin, a Transforming Growth Factor beta1 (TGFβ1) co-receptor. Here we show that TGFβ1 down-regulates PRH expression in prostate cells and thereby up-regulates cell migration.Material and methodsNormal immortalised prostate epithelial cells (PNT2-C2 cells) and prostate adenocarcinoma cells (PC3 cells) were treated with TGFβ1 (5 ng/ml) or vehicle for 48 hours. mRNA expression was then assayed using quantitative PCR. Protein expression was assayed using western blotting and immunofluorescence microscopy. Cell migration was analysed using chemotaxis assays. pSMAD3 binding was examined using quantitative chromatin immunoprecipitation assays.Results and discussionsThe treatment of immortalised prostate epithelial cells and prostate cancer cells with TGFβ1 increases cell migration. In immortalised prostate epithelial cells this is accompanied by morphological changes suggestive of an epithelial to mesenchymal transition (EMT). In both immortalised prostate epithelial cells and prostate cancer cells TGFβ1 treatment results in the down-regulation of E-cadherin expression and the up-regulation of SNAIL expression, two established hallmarks of EMT. In both cell lines TGFβ1 treatment results in increased levels of the TGFβ1 effector pSMAD3, increased binding of pSMAD3 to the PRH/HHEX gene, and down-regulation of PRH expression at both the protein and mRNA level. Interestingly, PRH over-expression increases E-cadherin levels and represses cell migration.ConclusionTGFβ1 treatment increases prostate cell migration in part at least by the down-regulation of PRH expression. PRH up-regulates E-cadherin expression and down-regulates cell migration suggesting that TGFβ1 alters E-cadherin levels and increases cell migration via its effects on PRH. Since PRH also regulates TGFβ1 signalling this creates a feedback loop that enables a more precise control of cell behaviour. Changes in PRH levels or activity during tumourigenesis disrupt this control mechanism.

Abstract 4485: VGLL3 is a novel outlier in African American prostate tumors and its expression is associated with a tumor suppressor phenotype in prostate cancer

Abstract Higher incidence, progression and mortality of prostate cancer (PCa) have been noted in African American (AA) men compared with Caucasian (CA) men. In fact AA race is an independent predictor of biochemical recurrence for all stage of PCa (Dr. Schaeffer's data analysis, n=1634 of AA patients). The etiology of PCa racial disparities is likely multi-factorial with increasing support of biologic contributors to these differences. By analyzing large cohort of PCa, we have demonstrated that AA PCa exhibits a unique spectrum of molecular subtypes. For example, AA men presented with PCa have a significant decrease in fusion proteins such as ERG, ETS and SPINK, with an enrichment of fusion unknown (FU; triple negative) cases. Given the increased mortality of PCa in AA populations and the enrichment of FU cases among AA tumors, there is an urgent need to define and explore individual outlier as race-based modifiers of disease aggressiveness. By utilizing a large multi-institutional cohort of prostate cancer cases of mixed racial composition, we have identified novel outliers enriched in AA among FU group by COPA outlier analysis.VGLL3 is one of the top outliers enriched in AA. VGLL3 positive cases are associated with low stage (less LNI, p=0.001; SVI, p<0.001; and EPE, p=0.009) and low grade disease (less Gleason >7, p=0.012) of higher protective phenotype of adverse cancer grade and stage. This preliminary work on VGLL3 expression suggests it may be associated with a tumor suppressor-like phenotype with reductions in expression with increasingly aggressive PCa. Studies of VGLL3 in cancer are limited and the role of VGLL3 in prostate has not been explored at all. The goal of this study is to investigate the role of VGLL3 as a novel outlier in AA prostate cancer that modulates disease aggressiveness. We found that mRNA expression levels of VGLL3 were lower in prostate cancer epithelial cells than in normal epithelial cells or stromal cells. By establishing and characterizing PCa models overexpressing VGLL3 in vitro and in vivo, our data demonstrate that VGLL3 significantly reduced migration and invasion of PCa cells and inhibited primary tumor cell growth. Our preliminary data also indicate VGLL3 inhibits PCa metastasis. Furthermore, VGLL3 seems to negatively affect both hippo and AR activity in PCa cells and these might be the mechanisms that VGLL3 inhibits tumor cell growth. These data is consistent with our clinical data suggesting tumor suppressive role of VGLL3 in advanced PCa. Citation Format: Hongjun Kang. VGLL3 is a novel outlier in African American prostate tumors and its expression is associated with a tumor suppressor phenotype in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4485.

Abstract 61: Obesity-induced T cell senescence contributes to prostate cancer progression

Abstract Obese men are at greater risk of developing an aggressive form of prostate cancer. The T cell population from obese subjects have shown signatures of cell senescence including shortened telomeres and elevated production of proinflammatory cytokines. The objective of our studies is to better understand the cellular and molecular basis for altered T cell anti-prostate tumor immunity in obesity, and whether this altered immune function contributes to the worse outcomes observed in obese patients. To investigate whether obesity induces a pro-inflammatory senescent phenotype in CD4+ T cells, splenic CD4+ T-cells from 10-week-old male C57Bl/6 mice were isolated and, either pre-stimulated or co-stimulated with anti CD3/CD28 and conditioned media (CM) from macrophages stimulated under obese or normo-weight conditions. After 24 hours conditioned media from these CD4+ T cells was collected, and their relative cytokine expression profile was analyzed. The CD28 receptor is expressed on CD4+ T cells and provides co-stimulatory signals required for T cell activation. CD28 activation results in phosphorylation of Akt. Lack of Akt phosphorylation in CD4+ T cells after CD3+/CD28+ stimulation indicates CD4+ T cell dysfunction and senescence. Fresh murine splenic CD4+ T cells were isolated and stimulated with anti CD3/CD28 under obese or control conditions. Akt1 phosphorylation levels were assessed using Western blot analysis. To determine if obese conditions modify the impact of CD4+ T cell dysfunction on prostate cancer cell signal transduction pathways, prostate cancer epithelial cells were exposed to obese or control CD4+ T-cells CM for 30 minutes and western blot analysis was used to evaluate the modulation of key pro- and anti-tumorigenic pathways. Matrigel Invasion chambers were used to determine if obesity-modified CD4+ T cell CM impacts the invasive capacity of prostate epithelial cells. Obese conditions suppressed the expression of IFN-γ and IL-2 in CD4+ T-cells. Data also suggests that T cells exposed to obese conditions suppress STAT1 activation in prostate cancer cells. Additionally, obese conditions up-regulated the expression of IL-6 and IL-10 in CD4+ T-cells when compared to control. Importantly our data also suggests that T cells exposed to obese conditions induce STAT3 activation in prostate cancer cells. Preliminary data also suggests that obesity-modified CD4+ T cell CM impacts the invasive capacity of prostate cancer cells. Obesity may induce CD4+ T cell dysfunction, limiting their tumor suppressing activity. Findings form this study could substantially advance fundamental understanding of the ways in which obesity alters T cells, thereby promoting uncontrolled tumor growth and mortality from prostate cancer. This study could also provide a framework for future translational research aimed at improving the effectiveness of therapies in obese patients. Citation Format: Alejandra De Angulo, Peyton Travis, Christopher Jolly, Linda deGraffenried. Obesity-induced T cell senescence contributes to prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 61.

PO-283 Characterisation of hTERT-immortalised prostate-derived stromal and epithelial cells: an authentic in vitro model for tumour microenvironment studies

IntroductionTumour development begins with mutational changes to the genetic makeup of a cell; tumour progression is not solely determined by the mutated cell, but also by the tumour’s microenvironment. Prostate cancer, a leading cancer diagnosed in men, has been determined to be highly influenced by its surrounding stroma, particularly fibroblasts. It has been demonstrated that cancer-associated prostate fibroblasts (CAFs) differ from normal-associated prostate fibroblasts (NAFs). However, human prostate cancer model systems have focused largely on prostate cancer epithelial cells. Currently, a need exists for a more physiologically relevant human cell model system to study prostate cancer progression within the context of its tumour microenvironment.Material and methodsIn this study, we characterised three prostate-derived cells: CAFs, NAFs, and prostate epithelial cells (PrEs); all three lines were immortalised by human telomerase reverse transcriptase (hTERT) alone and selected with puromycin. The selected clones grew continuously in culture for more than 40 population doublings in our hands.Results and discussionsOur data shows that the hTERT-immortalised CAFs proliferate faster than the NAFs; in addition, both CAFs and NAFs express fibroblast markers such as TE7 and alpha smooth muscle actin (a-SMA), while neither cell line expresses epithelial markers such as CK14. Both CAFs and NAFs also express elevated levels of a-SMA upon TGF-b stimulation. All three prostate-derived cells weakly express the prostate specific marker AR, and show similar markers staining after long time passaging. Importantly, conditioned media collected from CAFs promotes tumour cell growth better than NAF conditioned media.ConclusionIn conclusion, CAFs, NAFs, and immortalised PrEs may provide a very valuable model system for the study of prostate cancer cell progression and tumour microenvironment studies.