RWPE-1 Cells Research Articles

Abstract 1595: HMGA2 induces EMT in prostate cancer cells and may be antagonized by camalexin

Abstract Prostate cancer is the most diagnosed cancer in men and the second leading cause of death in the United States. African American men have the highest incidence and mortality rates of prostate cancer compared to any other race. Epithelial-mesenchymal transition (EMT) plays a critical role in cancer progression and metastasis. Mesenchymal cells are migratory, invasive, and more resistant to apoptosis. Reactive Oxygen Species (ROS) has been shown to promote EMT. High mobility group A (HMGA2) is a non-histone protein that is highly expressed during the embryogenesis, whereas the gene expression is very low or absent during adulthood. Recent studies have been reported an overexpression of HMGA2 protein in malignant cancers. Loss of Let-7 miRNA (repressor of HMGA2) has been found to induce EMT via upregulation of HMGA2 in prostate cancer. There has been no link between ROS and HMGA2. We have reported that camalexin, a 3-thizol-2-yl-indole, may be a candidate treatment for aggressive prostate cancer cells by ROS-mediated apoptosis. The study demonstrated that treating the prostate cancer cell with camalexin increased ROS levels which contributed to decreased cell proliferation and increased apoptosis. We hypothesize that HMGA2 may regulate EMT in part by inducing ROS and that camalexin may antagonize HMGA2 signaling. We analysed HMGA2 and EMT marker expression in a panel of prostate cancer cell lines by western blot analysis. We also transiently and stably overexpressed wild-type HMGA2 and mutant HMGA2 (missing Let-7 binding site) in LNCaP cells. We measured ROS levels using DCFDA dye that detects hydrogen peroxide. We treated ARCaP-M (mesenchymal) cells with different concentrations of camalexin to analyse HMGA2 expression. Our results showed that HMGA2 is highly expressed in aggressive prostate cancer cell lines (C4-2, ARCaP, E006AA) as compared to RWPE1 and LNCaP cells. The transient overexpression of HMGA2 in LNCaP cells decreased E-cadherin more markedly than with mutant HMGA2 but did not show any changes in ROS. We will repeat this experiment with stable clones from HMGA2 overexpression. ARCaP-M cells treated with camalexin induced ROS and decreased HMGA2 expression. We are currently performing in vivo studies using ARCaP-M cells injected subcutaneously into nude mice followed by treatment with camalexin after the tumor grows to 50 mm3. In conclusion, HMGA2 promotes EMT, even more markedly if its Let-7 suppressor binding site is eliminated, and camalexin may target HMGA2 to decrease prostate cancer progression. GRANT SUPPORT: 1P20MD002285; 8G12MD007590 Citation Format: Ohuod A. hawsawi, Basil Smith, Jodi Dougan, Liza J. Burton, Valerie A. Odero-Marah. HMGA2 induces EMT in prostate cancer cells and may be antagonized by camalexin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1595.

Abstract 1091: miR-125 family of miRNAs mediates prostate cancer cell proliferation and migration

Abstract Background: Prostate cancer is the second most common malignancy in American men and causes ∼27540 deaths per annum. microRNAs (miRNAs) are a class of short non-coding RNAs that regulate the expression of protein-coding genes via either mRNA degradation or translational repression. These small molecules are known to be aberrantly expressed in prostate cancer (PCa) and possess both tumor suppressive and oncogenic properties. miR-125 is a highly conserved miRNA family comprised of three homolog members hsa-miR-125a, hsa-miR-125b-1, and hsa-miR-125b-2. Members of this family have been attributed both disease suppressing and promoting functions in certain contexts however their role in PCa has yet to be fully elucidated. Methods: miRNA expression was evaluated in a publicly available dataset (GSE21032) comprised of 28 normal, 98 primary PCa and 13 metastasis samples. miR-125 expression was assessed in a panel of 8 prostate cell lines comprising PrEC prostate epithelial cells, RWPE-1, BPH1, DU145, LNCaP, C4-2B, PC3, and PC3-2M cell lines using the Qiagen miScript system. MirWalk was used to identify predicted targets of the miRNAs. miR-125 expression was transiently inhibited in LNCaP, C4-2B, PC3, and PC3-2M cell lines using miScript miRNA inhibitors and miRNA and target mRNA expression, cell proliferation and migration were measured post-transfection. Results: Analysis of human data revealed miR-125a-3p was significantly up-regulated in metastasis samples compared to both primary PCa and normal tissues (log fold change (FC) 1.743, p-value 9.01E-08 logFC = 1.536 p-value = 8.80E-08 respectively). Next, we validated miR-125 family member expression in prostate cancer cell lines compared to PrEC and RWPE-1 prostate epithelial cells. Most notably, we observed that miR-125b-2 expression was significantly higher in all prostate cancer cell lines. Inhibition of miR-125b-2, significantly reduced the proliferation of the LNCaP, C4-2B, and PC3-2M cell lines. Moreover, inhibition of this miRNA significantly impaired prostate cancer cell migration by 43.8% (p-value = 0.003). Using MirWalk, we examined potential targets for regulation by miR-125b-2 and identified the potent tumor suppressor Quaking family as top hit. Our results show that inhibition of miR-125b-2 significantly enhanced the expression of Quaking family members (1.79 fold). Conclusions: Our results demonstrate that the miR-125 family of miRNAs plays a role in regulating prostate cancer cell proliferation and migration, potentially by mitigating Quaking activity. Citation Format: Sinead T. Aherne, Fiona O’Neill, Stephen F. Madden, Martin Clynes, Conor C. Lynch. miR-125 family of miRNAs mediates prostate cancer cell proliferation and migration. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1091.

Abstract 2615: Dietary flavonoid fisetin increases abundance of high-molecular-mass hyaluronan conferring resistance to prostate oncogenesis

Abstract With an estimated 220,800 new cases and 27,450 deaths in 2015, prostate cancer (PCa) continues to be the second leading cause of cancer related deaths in men in the US alone. Accumulation and turnover of extracellular matrix (ECM) is a hallmark of tissue injury, repair and remodeling in human diseases including PCa. Hyaluronan (HA), an extracellular glycosaminoglycan, is a major component of the ECM and plays an important role in regulating reepithelization, tissue healing and wound repair. Elevated HA levels have been shown to be associated with aggressiveness and disease progression in certain cancer types including PCa. Biological responses triggered by HA mainly depend on the HA polymer length. High molecular mass (HMM)-HA represses mitogenic signaling and has anti-inflammatory properties while low molecular mass (LMM)-HA is pro-angiogenic and promotes proliferation and inflammation. Diet-derived agents, such as flavonoids, are of particular interest for cancer chemoprevention as they offer a relatively favorable safety profile. Using HPLC-MS analysis, we identified HA as a novel target of the dietary flavonoid fisetin in tumor xenografts of human PCa NB11 and NB26 cells. Fisetin treatment decreased HA levels in PCa cells both in-vitro (40μM:40h) and in-vivo (1mg/animal). We then evaluated the effect of intraperitoneal administration of fisetin on HA size accumulation and prostate carcinogenesis using the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Using the combination of Size Exclusion Chromatography (SEC) with Multi-Angle Laser Light Scattering (MALS) analysis that overcomes limitations of column calibration, we evaluated intracellular and extracellular molar mass distribution of HA in fisetin treated cell culture and animal models. We observed lower levels of HMM-HA and higher levels of pro-angiogenic LMW-HA fragments in human PCa PC3 cells. In contrast, we found that fisetin treatment increased HMM-HA and reduced LMM-HA levels in PCa cells. Remarkably, the HA size profile of fisetin treated cells resembled those of normal RWPE1 cells, which predominantly exhibited higher abundance of anti-angiogenic HMM-HA and no detectable LMW-HA. Likewise, secreted HA in fisetin treated PCa cells comprised predominantly of LMM-HA, suggesting greater accumulation of anti-angiogenic HMM-HA within the cells. Molar mass analysis of fisetin treated TRAMP tumor tissues showed an abundance of HMM-HA and reduced levels of LMM-HA when compared to untreated controls. Similarly, secreted HA in fisetin treated TRAMP animals exhibited increased pool of pro-angiogenic LMM-HA, suggesting greater accumulation of anti-angiogenic HMM-HA within the tissues. Our findings suggest that fisetin confers resistance to PCa oncogenesis by increasing the abundance of anti-angiogenic HMM-HA which is associated with delayed disease progression. Citation Format: Rahul K. Lall, Deeba N. Syed, Mohammad Imran Khan, Vaqar M. Adhami, Yuansheng Gong, John A. Lucey, Hasan Mukhtar. Dietary flavonoid fisetin increases abundance of high-molecular-mass hyaluronan conferring resistance to prostate oncogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2615.

SPOCK1 promotes tumor growth and metastasis in human prostate cancer.

Prostate cancer is the most diagnosed noncutaneous cancer and ranks as the second leading cause of cancer-related deaths in American males. Metastasis is the primary cause of prostate cancer mortality. Survival rate is only 28% for metastatic patients, but is nearly 100% for patients with localized prostate cancers. Molecular mechanisms that underlie this malignancy remain obscure, and this study investigated the role of SPARC/osteonectin, cwcv, and kazal-like domain proteoglycan 1 (SPOCK1) in prostate cancer progression. Initially, we found that SPOCK1 expression was significantly higher in prostate cancer tissues relative to noncancerous tissues. In particular, SPOCK1 expression was also markedly high in metastatic tissues compared with nonmetastatic cancerous tissues. SPOCK1 expression knockdown by specific short hairpin RNA in PC3 cells was significantly inhibited, whereas SPOCK1 overexpression in RWPE-1 cells promoted cell viability, colony formation in vitro, and tumor growth in vivo. Moreover, the SPOCK1 knockdown in PC3 cells was associated with cell cycle arrest in G0/G1 phase, while the SPOCK1 overexpression in RWPE-1 cells induced cell cycle arrest in S phase. The SPOCK1 knockdown in PC3 cells even increased cell apoptosis. SPOCK1 modulation was also observed to affect cancerous cell proliferation and apoptotic processes in the mouse model of prostate cancer. Additionally, the SPOCK1 knockdown decreased, whereas the SPOCK1 overexpression increased cell migration and invasion abilities in vitro. Injection of SPOCK1-depleted PC3 cells significantly decreased metastatic nodules in mouse lungs. These findings suggest that SPOCK1 is a critical mediator of tumor growth and metastasis in prostate cancer.

The effects of metformin and simvastatin on the growth of LNCaP and RWPE-1 prostate epithelial cell lines

The anti-diabetic drug metformin and cholesterol-lowering statins inhibit prostate cancer cell growth in vitro and have been linked with lowered risk of prostate cancer in epidemiological studies. We evaluated the effects of these drugs on cancerous and non-cancerous prostate epithelial cell lines. Cancer (LNCaP) and normal (RWPE-1) prostate epithelial cell lines were treated with pharmacologic concentrations of metformin and simvastatin alone and in combinations. Relative changes in cell number were measured with crystal violet staining method. Drug effects on apoptosis and cell cycle were measured with flow cytometry. We also measured changes in the activation and expression of a set of reported target proteins of metformin and statins with Western blotting. Metformin decreased the relative cell number of LNCaP cells by inducing G1 cell cycle block, autophagy and apoptosis, and slightly increased cytosolic ATP levels, whereas RWPE-1 cells were resistant to metformin. However, RWPE-1 cells were sensitive to simvastatin, which induced G2 cell cycle block, autophagy and apoptosis, and increased cytosolic ATP levels in these cells. Combination of metformin and simvastatin synergistically decreased cytosolic ATP levels, increased autophagy and instead of apoptosis, induced necrosis in LNCaP cells. Synergistic effects were not observed in RWPE-1 cells. These results suggest, that prostate cancer cells may be more vulnerable to combined growth-inhibiting effects of metformin and simvastatin compared to normal cells. The data presented here provide evidence for the potency of combined metformin and statin, also at pharmacologic concentrations, as a chemotherapeutic option for prostate cancer.

PlncRNA-1 induces apoptosis through the Her-2 pathway in prostate cancer cells.

To determine whether PlncRNA-1 induces apoptosis in prostate cancer cells through the Her-2 pathway. The expression of PlncRNA-1, Her-2, and related cyclin proteins in 23 cases of prostate cancer and adjacent normal tissues was analyzed and compared. LNCaP cells were divided into a control group and an LNCaP-PlncRNA-1-siRNA experimental group. Normal prostate RWPE-1 cells were divided into an RWPE-1 control group and an RWPE-1-PlncRNA-1 experimental group. After PlncRNA-1 silencing and overexpression, changes in Her-2 and cyclinD1 expression levels were detected both in vivo and in vitro. In prostate cancer tissues, Her-2 and PlncRNA-1 were highly expressed and significantly correlated. In LNCaP cells, the expression of Her-2 and cyclinD1 decreased following the downregulation of PlncRNA-1 as assessed by real-time PCR and Western blotting. In RWPE-1 cells, the expression of Her-2 and cyclinD1 increased following PlncRNA-1 overexpression. Flow cytometry revealed that the proportion of LNCaP cells in G2/M phase was significantly increased after PlncRNA-1 silencing and that the proportion of RWPE-1 cells in G2/M phase was significantly decreased after PlncRNA-1 overexpression. Furthermore, animal experiments validated these results. In conclusion, in prostate cancer, PlncRNA-1 regulates the cell cycle and cyclinD1 levels and can also regulate proliferation and apoptosis in prostate cancer cells through the Her-2 pathway.

Oxidative phosphorylation and mitochondrial function differ between human prostate tissue and cultured cells.

Altered mitochondrial metabolism plays a pivotal role in the development and progression of various diseases, including cancer. Cell lines are frequently used as models to study mitochondrial (dys)function, but little is known about their mitochondrial respiration and metabolic properties in comparison to the primary tissue of origin. We have developed a method for assessment of oxidative phosphorylation in prostate tissue samples of only 2 mg wet weight using high-resolution respirometry. Reliable protocols were established to investigate the respiratory activity of different segments of the mitochondrial electron transfer system (ETS) in mechanically permeabilized tissue biopsies. Additionally, the widely used immortalized prostate epithelial and fibroblast cell lines, RWPE1 and NAF, representing the major cell types in prostate tissue, were analyzed and compared to the tissue of origin. Our results show that mechanical treatment without chemical permeabilization agents or sample processing constitutes a reliable preparation method for OXPHOS analysis in small amounts of prostatic tissue typically obtained by prostate biopsy. The cell lines represented the bioenergetic properties of fresh tissue to a limited extent only. Particularly, tissue showed a higher oxidative capacity with succinate and glutamate, whereas pyruvate was a substrate supporting significantly higher respiratory activities in cell lines. Several fold higher zinc levels measured in tissue compared to cells confirmed the role of aconitase for prostate-specific metabolism in agreement with observed respiratory properties. In conclusion, combining the flexibility of cell culture models and tissue samples for respirometric analysis are powerful tools for investigation of mitochondrial function and tissue-specific metabolism.

MP36-16 THE EXPRESSION OF BETA-DEFENSIN 131 PROMOTES INNATE IMMUNITY IN HUMAN PROSTATE EPITHELIAL CELLS

You have accessJournal of UrologyInfections/Inflammation/Cystic Disease of the Genitourinary Tract: Prostate & Genitalia II1 Apr 2016MP36-16 THE EXPRESSION OF BETA-DEFENSIN 131 PROMOTES INNATE IMMUNITY IN HUMAN PROSTATE EPITHELIAL CELLS Jung Hoon Kim, Kyeoung-Hwa Kim, Hae Jong Kim, Jaekhyouk Lee, Soon Chul Myung, Jae Duck Choi, and Seung Hyun Ahn Jung Hoon KimJung Hoon Kim More articles by this author , Kyeoung-Hwa KimKyeoung-Hwa Kim More articles by this author , Hae Jong KimHae Jong Kim More articles by this author , Jaekhyouk LeeJaekhyouk Lee More articles by this author , Soon Chul MyungSoon Chul Myung More articles by this author , Jae Duck ChoiJae Duck Choi More articles by this author , and Seung Hyun AhnSeung Hyun Ahn More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1650AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Previously, we found that ß-defensin 131 (DEFB131), an antimicrobial peptide, is upregulated in the human prostate epithelial cell line, RWPE-1, upon stimulation with lipoteichoic acid (LTA), a gram-positive bacterial component, compared to untreated RWPE-1 cells, using the Illumina HumanHT-12 microarray. In the current study, we aimed to investigate the role of DEFB131 in RWPE-1 cells during bacterial infection. METHODS We examined intracellular signaling pathways and nuclear responses in RWPE-1 cells that contribute to DEFB131 gene induction upon stimulation with LTA. Chromatin immunoprecipitation was performed to determine whether NF-?B directly binds to the DEFB131 promoter after LTA stimulation in RWPE-1 cells. RESULTS We found that DEFB131 expression was induced by LTA stimulation through TLR2 and p38MAPK/NF-?B activation, which was evaluated by phosphorylation of both p38MAPK and I?Ba. We also found that SB203580 and Bay11-7082, inhibitors of p38MAPK and NF-?B, respectively, suppressed LTA-induced DEFB131 expression. A chromatin immunoprecipitation assay showed that NF-?B directly binds to the DEFB131 promoter, suggesting that NF-?B is a direct regulator, and is necessary for LTA-induced DEFB131 expression in RWPE-1 cells. Interestingly, upon DEFB131 overexpression in RWPE-1 cells, mRNA accumulation and protein secretion of cytokines (IL-1a, IL-1ß, IL-6, and IL-12a) and chemokines (CCL20, CCL22, and CXCL8) were significantly enhanced. In addition, DEFB131-transfected RWPE-1 cells markedly induced chemotactic activity in THP-1 monocytes. CONCLUSIONS We concluded that DEFB131 induces cytokine and chemokine upregulation through the TLR2/NF-?B signaling pathway in RWPE-1 cells during bacterial infection and promotes innate immunity. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e495 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Jung Hoon Kim More articles by this author Kyeoung-Hwa Kim More articles by this author Hae Jong Kim More articles by this author Jaekhyouk Lee More articles by this author Soon Chul Myung More articles by this author Jae Duck Choi More articles by this author Seung Hyun Ahn More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MicroRNA alteration and putative target genes in high-grade prostatic intraepithelial neoplasia and prostate cancer: STAT3 and ZEB1 are upregulated during prostate carcinogenesis.

We aimed to identify alteration of cancer-related miRNAs in HGPIN and PCa, and to investigate the clinical implications of HGPIN as a precancerous lesion of PCa. Clinicopathologic analysis based on the status of HGPIN was performed in 388 patients who received radical prostatectomy between January 2005 and December 2008 in Severance Hospital. Among them, 10 paired HGPIN and PCa were prepared to perform miRNA microarray and quantitative real-time PCR. Fifty-two prostatectomy specimens were used to further validation of protein expression that was assessed by immunohistochemical staining (IHC) in matched non-neoplastic prostatic tissue (NPT), HGPIN, and PCa. Functional analysis was performed using a prostate normal cell line (RWPE-1) and two prostate cancer cell lines (LNCaP, PC-3) for comparison of expression of miR-155 and STAT3 mRNA before and after treatment of miR-155 mimetics/antagomir into each cell line. Patients with HGPIN had significantly less lymphovascular invasion, less lymph node metastasis, lower tumor volume, lower Gleason score, lower incidence of death, and longer overall survival compared to patients without HGPIN. MiR-155, miR-210, miR-153, and miR-200c were downregulated in HGPIN and PCa in common, compared to NPT. As putative target mRNAs, mRNA expression level of STAT3, ZEB1, and BACH1 was increased in PCa and HGPIN compared to NPT. mRNA expression level of ephrin-A3 was increased in PCa compared to NPT, and FGFRL1 was decreased in PCa compared to HGPIN and NPT. Protein expression assessed by IHC showed correlated results in STAT3, ZEB1, and ephrin-A3. Moreover, STAT3 and ZEB1 increased in a stepwise manner, from NPT to PCa. Treatment of miR-155 antagomir increased STAT3 mRNA expression in RWPE-1 cells, whereas treatment of miR-155 mimetics into PC-3 cells significantly decreased STAT3 expression. STAT3 and ZEB1 could be the key molecules altered at the early stages of carcinogenesis, especially in HGPIN. Prostate 76:937-947, 2016. © 2016 Wiley Periodicals, Inc.

Abstract B34: Secreted microRNAs from prostate cancer cells: Novel therapeutic targets

Abstract Introduction: More than 80% of men with aggressive prostate cancer (PCa) develop metastatic disease and skeletal complications leading to a dramatic reduction in quality of life. There is a deficit in effective biomarkers predicting disease progression, response to treatment, or metastasis at early stages when intervention would be possible. A critical need exists for novel therapeutic targets to prevent PCa metastasis to bone. MicroRNAs (miRNA) are informative biomarkers, as they circulate, are easily detected in human serum, and are coupled to targets reflecting deregulated pathways. Secreted miRNAs may be bound to proteins, membrane-encapsulated, or free RNA molecules and can act as intercellular signaling molecules. Here we tested the hypothesis that miRNAs secreted from prostate cancer cells in the primary tumor function locally and at distal sites to alter the normal tissue environment and promote PCa. Objective: Use global miRNA and gene expression analysis to 1) define cohorts of miRNAs secreted from prostate cancer cells representing different disease stages, 2) identify expression levels of their target mRNAs, and 3) provide insight into mechanisms of secreted miRNA activity in promoting prostate cancer tumor growth and metastasis. Methods: RNA was isolated from normal prostate epithelial (RWPE-1), early stage, androgen responsive PCa (LNCaP), and late stage, androgen insensitive PCa (PC-3) cell lines and from their conditioned media. Global expression of both secreted and extracellular miRNAs were interrogated using Affymetrix miRNA v4.0 microarrays (miRBase v20). Stranded RNA-seq was performed in parallel to identify differential expression of miRNA target mRNAs. We utilized the RNA-seq data to limit predicted mRNAs to those having reciprocal expression changes to their targeting miRNA. Enriched biological pathways of candidate miRNAs were identified for these predicted mRNA targets. Results: To identify secreted miRNAs that are candidates for promoting prostatic tumor growth, cancer progression, and metastatic bone disease, we compared differentially expressed miRNAs between PCa cell lines and their conditioned media. While numerous miRNAs are found to be both secreted and expressed robustly in the cell layer, we find unique subsets of miRNAs in each cell line that are either preferentially secreted or retained within the cell. We examined putative mRNA targets of these miRNAs in our gene expression data and established reciprocal mRNA-miRNA relationships. The miRNAs were found to be associated with pathways known to play a role in PCa and metastatic bone disease including ErbB, Wnt, TGF-beta, and MAPK signaling, as well as previously unassociated biological processes. Functional in vitro assays were performed for several candidate miRNAs that were overexpressed or inhibited in normal prostate epithelial RWPE-1 and highly metastatic PC-3 cell lines. Phenotypic changes, including motility, invasion, migration, and proliferation, were monitored. As an example, inhibition of miR-30a-5p, an established tumor suppressor, increases the rate at which RWPE-1 and PC-3 cells migrate to heal a cell layer scratch while overexpression prevents wound closure in RWPE-1 cells. In vivo orthotopic experiments to assay the ability of candidate miRNAs to promote local and metastatic PCa tumor growth are ongoing. Conclusion: Our combined data identify, for the first time, miRNAs secreted from prostate tumor cells that promote metastatic events of prostate cancer. Several of these miRNAs have been reported to be elevated in PCa patient serum and can provide diagnostic/prognostic biomarkers of tumor development, response to treatment, and recurrence. Our findings link these miRNAs to deregulated cellular pathways for potential intervention of prostate cancer progression. Citation Format: Nicholas H. Farina, Cody J. Callahan, Coralee E. Tye, Joseph R. Boyd, Gary S. Stein, Janet L. Stein, Jane B. Lian. Secreted microRNAs from prostate cancer cells: Novel therapeutic targets. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr B34.

Trichomonas vaginalis induces IL-1β production in a human prostate epithelial cell line by activating the NLRP3 inflammasome via reactive oxygen species and potassium ion efflux.

Trichomonas vaginalis is a sexually transmitted protozoan parasite that causes vaginitis in women, and urethritis and prostatitis in men. IL-1β is synthesized as immature pro-IL-1β, which is cleaved by activated caspase-1. Caspase-1 is, in turn, activated by a multi-protein complex known as an inflammasome. In this study, we investigated the inflammatory response of a prostate epithelial cell line (RWPE-1) to T. vaginalis and, specifically, the capacity of T. vaginalis to activate the NLRP3 inflammasome. RWPE-1 cells were stimulated by live T. vaginalis, and subsequent expression of pro-IL-1β, IL-1β, NLRP3, ASC and caspase-1 was determined by real-time PCR and Western blotting. IL-1β and caspase-1 production was also measured by ELISA. To evaluate the effects of NLRP3 and caspase-1 on IL-1β production, the activated RWPE-1 cells were transfected with small interfering RNAs to silence the NLRP3 and caspase-1 genes. Activation of the NLRP3 inflammasome was observed by fluorescence microscopy. Intracellular reactive oxygen species (ROS) were evaluated by spectrofluorometry. When RWPE-1 cells were stimulated with live T. vaginalis, the mRNA and protein expression of IL-1β, NLRP3, ASC, and caspase-1 increased. Moreover, silencing of NLRP3 and caspase-1 attenuated T. vaginalis-induced IL-1β secretion. The NADPH oxidase inhibitor DPI and high extracellular potassium ion suppressed the production of IL-1β, caspase-1, and the expression of NLRP3 and ASC proteins. The specific NF-κB inhibitor, Bay 11-7082, inhibited IL-1β production, and also inhibited the production of caspase-1, ASC and NLRP3 proteins. T. vaginalis induces the formation of the NLRP3 inflammasome in human prostate epithelial cells via ROS and potassium ion efflux, and this results in IL-1β production. This is the first evidence for activation of the NLRP3 inflammasome in the inflammatory response by prostate epithelial cells infected with T. vaginalis. Prostate 76:885-896, 2016. © 2016 Wiley Periodicals, Inc.

N-acetylcysteine Ameliorates Prostatitis via miR-141 Regulating Keap1/Nrf2 Signaling.

Chronic prostatitis was the most common type of prostatitis and oxidative stress was reported to be highly elevated in prostatitis patients. In this study, we determined the effect of N-acetylcysteine (NAC) on prostatitis and the molecular mechanism involved in it. Male Sprague-Dawley rats were divided into three groups: control group (group A, n = 20), carrageenan-induced chronic nonbacterial prostatitis (CNP) model group (group B, n = 20), and carrageenan-induced CNP model group with NAC injection (group C, n = 20). Eye score, locomotion score, inflammatory cell count, cyclooxygenase 2 (COX2) expression, and Evans blue were compared in these three groups. The expression of miR-141 was determined by quantitative real-time PCR (qRT-PCR). Moreover, protein expressions of Kelch-like ECH-associated protein-1 (Keap1) and nuclear factor erythroid-2 related factor 2 (Nrf2) and its target genes were examined by Western blot. Luciferase reporter assay was performed in RWPE-1 cells transfected miR-141 mimic or inhibitor and the plasmid carrying 3'-UTR of Keap1. The value of eye score, locomotion score, inflammatory cell count, and Evans blue were significantly decreased in group C, as well as the expression of COX2, when comparing to that of group B. These results indicated that NAC relieved the carrageenan-induced CNP. Further, we found that NAC increased the expression of miR-141 and activated the Keap1/Nrf2 signaling. Luciferase reporter assay revealed that miR-141 mimic could suppress the activity of Keap1 and stimulate the downstream target genes of Nrf2. In addition, miR-141 inhibitor could reduce the effect of NAC on prostatitis. NAC ameliorates the carrageenan-induced prostatitis and prostate inflammation pain through miR-141 regulating Keap1/Nrf2 signaling.

Fibroblasts prolong serum prostate-specific antigen decline after androgen deprivation therapy in prostate cancer

In patients with prostate cancer (PCa), serum prostate-specific antigen (PSA) is a useful marker for evaluating the effects of androgen deprivation therapy (ADT). Intuitively, most urologists expect that a more rapid PSA decline in response to ADT would be positively associated with extended survival. Recently, we have reported that prolonged gradual serum PSA decline after ADT is strongly associated with favorable prognosis in PCa patients, however, the mechanism remains unknown. We investigated the role of fibroblasts in serum PSA decline after ADT. We performed in vitro experiments using androgen-sensitive, androgen receptor (AR)-positive prostate epithelial cell lines (LNCaP, 22Rv1, and RWPE-1 cells), commercially available prostate stromal cells (PrSC), and primary cultures of prostate fibroblasts (pcPrFs). In LNCaP and 22Rv1 cells, PSA production was increased by co-culture with fibroblasts under androgen-deprived conditions. In an in vivo model using LNCaP cells, serum PSA declined rapidly after ADT becoming undetectable within 14 days in mice inoculated with LNCaP cells alone. In contrast, when LNCaP cells were co-inoculated with fibroblasts, serum PSA levels were still high on 14 days post ADT and did not drop to undetectable levels until 21 days post ADT. Tumor volumes and Ki67 labeling indices were not altered between days 14 and 21 post ADT in mice inoculated with LNCaP cells; however, those in mice inoculated with LNCaP cells plus fibroblasts decreased gradually. PSA protein was detected in all tumors on 21 days post ADT by immunohistochemical staining. Microvessel densities were higher on 14 days post ADT for tumors from mice inoculated with LNCaP cells plus fibroblasts as compared with LNCaP cells alone. In summary, co-inoculation of fibroblasts with LNCaP cells prolonged serum PSA decline after ADT and enhanced the efficacy of ADT. Prolonged serum PSA decline may indicate the presence of protective fibroblasts that preserve the AR dependence of PCa cells, improving treatment efficacy.

Abstract B04: Cohesin complex subunit (stromal antigen1): Potential molecular determinant of racial disparities in prostate cancer

Abstract Purpose: The overarching goal is to determine the molecular cause of racial disparities in Prostate Cancer (PCa) and exploit its clinical implications. Background: African Americans (AA) have two-thirds higher risk of being diagnosed with PCa with mortality rate twice as high compared to Whites. Evidence supports an earlier transformation to clinically significant PCa in AA. While PCa incidence has declined in the last decade, the racial disparity has not narrowed. Even the improved healthcare, socio-economic/lifestyle factors does not seem to benefit this disparity greatly. Thus, it is evocative that certain biological factors may hold crucial links, therefore necessitating emerging biomarkers for valuable risk stratification. We have recently provided a sensitive/specific optical biomarker as an indicator of field carcinogenesis, segregating patients with indolent vs aggressive PCa. A susceptible genetic background or a field effect is very vital for tumor initiation, allowing accumulation of mutations in neoplastic lesions leading to genomic instability. On these lines, recent studies have demonstrated novel role of cohesion complex genes driving aneuploidy and tumorigenesis. Intriguingly, our investigations in human bio-specimens revealed a consistent downregulation of a putative cohesion complex subunit, Stromal Antigen1 (SA1), in PCa progression. We hypothesized that “SA1 aberrations may serve as an indicator of genomic filed carcinogenesis, providing insights into the biological mechanisms for racial disparities in PCa”. For this, we aimed to characterize SA1 expression in PCa initiation/progression in AA vs Whites, primarily focusing on its relevance with the widely recognized signaling axis in PCa, (pAKT/PI3K), and androgen responsiveness, since both are disproportionately prevalent among AA. Methods: To characterize SA1 levels in PCa field carcinogenesis for AA vs Whites, immunohistochemistry (IHC) for SA1 and PI3K was performed on histologically normal biopsies collected from racially-distinct Gleason6-7 PCa patients undergoing prostactomy. Further, we investigated by IHC the SA1 expression/sub-cellular localization in PTEN (tumor suppressor gene that negatively regulates AKT/PI3K) PCa transgenic mice model. The findings were corroborated in cell-line models using PCa cells (PC3 and DU145) and prostate epithelial cells (RWPE1) by inhibitor studies (for PI3K(LY-294002), nuclear exportin CRM1(LeptomycinB)) via immunofluorescence/immunoblotting. Results: Interestingly, prostate biopsies from AA demonstrated amplified PI3K levels and attenuated SA1 expression with significantly higher cytoplasmic/nuclear ratio compared to Whites, indicating a localization-allied role of SA1. Additionally, SA1 demonstrated altered expression/localization wrt PTEN milieu: nuclear in benign prostatic tissues of PTEN+/+ mice, whereas relatively less and predominantly cytoplasmic in tumor tissues of PTEN-/- mice. Further, PC3 and DU145 PCa cells demonstrated lesser overall SA1 expression with cytoplasmic co-localization with CRM1, whereas in RWPE1 cells SA1 was localized to the nucleus/nuclear membrane. The mechanistic substantiation by antagonist studies revealed significant blocking (~2fold) of CRM1-mediated nuclear-cytoplasmic SA1 shuttling in PC3 cells with LY+LeptomycinB treatment. Moreover, by transiently downregulating SA1, substantial induction of androgen receptor (AR) was noted in AR-negative PC3 cells. Conclusions: Overall, our studies provide a greater role of pAKT/PI3K/PTEN and CRM1 in SA1 mislocalization, facilitating PCa field carcinogenesis, with potential implications in PCa aggressiveness and therapeutic resistance, the two major aspects of poor clinical outcome in AA patients. Our findings may lead to the discovery of novel biomarkers to identify high-risk AA men potentially providing druggable targets. Citation Format: Navneet Momi, Charles B. Brendler, Hemant K. Roy. Cohesin complex subunit (stromal antigen1): Potential molecular determinant of racial disparities in prostate cancer. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B04.

Synthetic Lethality of a Novel Small Molecule Against Mutant KRAS-Expressing Cancer Cells Involves AKT-Dependent ROS Production

We recently reported the death-inducing activity of a small-molecule compound, C1, which triggered reactive oxygen species (ROS)-dependent autophagy-associated apoptosis in a variety of human cancer cell lines. In this study, we examine the ability of the compound to specifically target cancer cells harboring mutant KRAS with minimal activity against wild-type (WT) RAS-expressing cells. HCT116 cells expressing mutated KRAS are susceptible, while the WT-expressing HT29 cells are resistant. Interestingly, C1 triggers activation of mutant RAS, which results in the downstream phosphorylation and activation of AKT/PKB. Gene knockdown of KRAS or AKT or their pharmacological inhibition resulted in the abrogation of C1-induced ROS production and rescued tumor colony-forming ability. We also made use of HCT116 mutant KRAS knockout (KO) cells, which express only a single WT KRAS allele. Exposure of KO cells to C1 failed to increase mitochondrial ROS and cell death, unlike the parental cells harboring mutant KRAS. Similarly, mutant KRAS-transformed prostate epithelial cells (RWPE-1-RAS) were more sensitive to the ROS-producing and death-inducing effects of C1 than the vector only expressing RWPE-1 cells. An in vivo model of xenograft tumors generated with HCT116 KRAS(WT/MUT) or KRAS(WT/-) cells showed the efficacy of C1 treatment and its ability to affect the relative mitotic index in tumors harboring KRAS mutant. These data indicate a synthetic lethal effect against cells carrying mutant KRAS, which could have therapeutic implications given the paucity of KRAS-specific chemotherapeutic strategies. Antioxid. Redox Signal. 24, 781-794.

Genetic and cellular studies highlight that A Disintegrin and Metalloproteinase 19 is a protective biomarker in human prostate cancer.

BackgroundProstate cancer is the second most frequently diagnosed cancer in men worldwide. Current treatments include surgery, androgen ablation and radiation. Introduction of more targeted therapies in prostate cancer, based on a detailed knowledge of the signalling pathways, aims to reduce side effects, leading to better clinical outcomes for the patient. ADAM19 (A Disintegrin And Metalloproteinase 19) is a transmembrane and soluble protein which can regulate cell phenotype through cell adhesion and proteolysis. ADAM19 has been positively associated with numerous diseases, but has not been shown to be a tumor suppressor in the pathogenesis of any human cancers. Our group sought to investigate the role of ADAM19 in human prostate cancer.MethodsADAM19 mRNA and protein levels were assessed in well characterised human prostate cancer cohorts. ADAM19 expression was assessed in normal prostate epithelial cells (RWPE-1) and prostate cancer cells (LNCaP, PC3) using western blotting and immunocytochemistry. Proliferation assays were conducted in LNCaP cells in which ADAM19 was over-expressed. In vitro scratch assays were performed in PC3 cells over-expressing ADAM19.ResultsImmunohistochemical studies highlighted that ADAM19 protein levels were elevated in normal prostate tissue compared to prostate cancer biopsies. Results from the clinical cohorts demonstrated that high levels of ADAM19 in microarrays are positively associated with lower stage (p = 0.02591) and reduced relapse (p = 0.00277) of human prostate cancer. In vitro, ADAM19 expression was higher in RWPE-1 cells compared to LNCaP cells. In addition, human ADAM19 over-expression reduced LNCaP cell proliferation and PC3 cell migration.ConclusionsTaken together, our immunohistochemical and microarray results and cellular studies have shown for the first time that ADAM19 is a protective factor for human prostate cancer. Further, this study suggests that upregulation of ADAM19 expression could be of therapeutic potential in human prostate cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2178-4) contains supplementary material, which is available to authorized users.

Lensfree diffractive tomography for the imaging of 3D cell cultures.

New microscopes are needed to help realize the full potential of 3D organoid culture studies. In order to image large volumes of 3D organoid cultures while preserving the ability to catch every single cell, we propose a new imaging platform based on lensfree microscopy. We have built a lensfree diffractive tomography setup performing multi-angle acquisitions of 3D organoid culture embedded in Matrigel and developed a dedicated 3D holographic reconstruction algorithm based on the Fourier diffraction theorem. With this new imaging platform, we have been able to reconstruct a 3D volume as large as 21.5 mm (3) of a 3D organoid culture of prostatic RWPE1 cells showing the ability of these cells to assemble in 3D intricate cellular network at the mesoscopic scale. Importantly, comparisons with 2D images show that it is possible to resolve single cells isolated from the main cellular structure with our lensfree diffractive tomography setup.

MiR-26b inhibits autophagy by targeting ULK2 in prostate cancer cells.

Autophagy is a catabolic process widely conserved among eukaryotes that permits the rapid degradation of unwanted proteins and organelles through the lysosomal pathway. The Serine/threonine protein kinase ULK2 (unc-51 like kinase 2) plays an important regulatory role in autophagy thanks to its involvement in mTOR-regulated-initiation and downstream ATG protein-related progression of this catabolic process. An increasing number of miRNAs have been found to modulate autophagy by targeting some ATG genes. In this study, we focus on the role of mir-26b in autophagy in prostate cancer (PCa) cells. We found that miR-26b inhibited autophagy in PC-3 and C4-2 cells, through down-regulation of ULK2 expression. Dual luciferase reporter assays showed that miR-26b binds the 3'UTR of ULK2, suggesting that ULK2 is a direct target of miR-26b. Real-time PCR and Western blot analysis confirmed that over-expression of miR-26b reduced ULK2 mRNA and protein levels. Our results showed also that miR-26b was down-regulated in LNCaP, DU145, C4-2 and PC-3 cells compared to the two normal prostate cells RWPE-1 and WPMY-1 except DU145 cells. This inversely correlates with ULK2 level in the same cell lines. Expression level of ULK2 in tissues microarray (TMA) of prostate cancer derived from 96 patients positively correlated with the pathologic stage of the patients (∗P < 0.05). Over-expression of ULK2 significantly reversed miR-26b-mediated autophagy inhibition. Taken together, our findings indicate that mir-26b inhibits autophagy through targeting ULK2 which is up-regulated in PCa.

Exosomes confer pro-survival signals to alter the phenotype of prostate cells in their surrounding environment.

Prostate cancer (PCa) is the most frequently diagnosed cancer in men. Current research on tumour-related extracellular vesicles (EVs) suggests that exosomes play a significant role in paracrine signaling pathways, thus potentially influencing cancer progression via multiple mechanisms. In fact, during the last decade numerous studies have revealed the role of EVs in the progression of various pathological conditions including cancer. Moreover, differences in the proteomic, lipidomic, and cholesterol content of exosomes derived from PCa cell lines versus benign prostate cell lines confirm that exosomes could be excellent biomarker candidates. As such, as part of an extensive proteomic analysis using LCMS we previously described a potential role of exosomes as biomarkers for PCa. Current evidence suggests that uptake of EV's into the local tumour microenvironment encouraging us to further examine the role of these vesicles in distinct mechanisms involved in the progression of PCa and castration resistant PCa. For the purpose of this study, we hypothesized that exosomes play a pivotal role in cell-cell communication in the local tumour microenvironment, conferring activation of numerous survival mechanisms during PCa progression and development of therapeutic resistance. Our in vitro results demonstrate that PCa derived exosomes significantly reduce apoptosis, increase cancer cell proliferation and induce cell migration in LNCaP and RWPE-1 cells. In conjunction with our in vitro findings, we have also demonstrated that exosomes increased tumor volume and serum PSA levels in vivo when xenograft bearing mice were administered DU145 cell derived exosomes intravenously. This research suggests that, regardless of androgen receptor phenotype, exosomes derived from PCa cells significantly enhance multiple mechanisms that contribute to PCa progression.

Abstract A16: Different epigenetic mechanisms involved in the regulation of SFRP1 gene in prostate cancer

Abstract Among men, prostate cancer (PCa)[1] is the second most common cancer and the fifth cause of death worldwide [2]. Androgens play an important role in the development of the disease. For advanced PCa the standard therapy is androgen depletion. However, after 2 or 3 years a high percentage of patients become resistant to this therapy and castration resistant prostate cancer (CRPC) is developed. There is no successful treatment for CRPC, leading into death [3, 4]. Wingless pathway (WNT) is aberrantly activated in several cancer types and in PCa it is involved in AR activity modulation, promoting cancer development [5]. Secreted Frizzled Related Protein 1 (SFRP1) is commonly known as a WNT inhibitor and generally proposed as a tumor suppressor gene. SFRP1 can exert its tumor suppressor activity by inhibiting the AR transcriptional activity [6]. Usually in cancer, SFRP1 mRNA is down regulated and this is associated to increased DNA methylation at its promoter region [7]. In contrast, in PCa there are different reports suggesting SFRP1 repression in absence of DNA methylation [8]. In this work we aimed to investigate two different epigenetic mechanisms for SFRP1 silencing in PCa. For the in vitro model, we used normal prostate cells RWPE-1 and PCa cell lines: LNCaP, 22RV1 and PC3. We confirmed the correlation between mRNA expression loss and the increased DNA methylation of SFRP1 only in PC3 cells, but not in LNCaP and 22RV1, were instead of DNA methylation we found enrichment of H3K27me3, the Polycomb Repressive Complex 2 (PRC2) mark. To correlate this findings, we analyzed the expression of SFRP1 in fresh frozen tissue samples, 11 primary PCa, 10 Benign Prostatic Hyperplasia (BPH) and 4 normal prostates (NP). We found SFRP1 downregulation in all PCa and BPH samples compared to Normal Prostate, but DNA methylation was increased only in PCa but not in BPH, this suggests a novel repressive mechanism for SFRP1 independent of DNA methylation. To see if AR has a role in regulating mRNA SFRP1 expression, we silenced AR with a siRNA, but no SFRP1 expression levels were modified, so we concluded AR was not directly involved in SFRP1 regulation. All these data together suggest different repression mechanisms for SFRP1, one depending on DNA methylation and another one depending on H3K27me3 PRC2 repressive mechanism, Androgen receptor is not directly involved in SFRP1 downregulation, so other factors might be involved in SFRP1 expression loss in PCa.