Androgen Dependence Research Articles

Sarcosine Up-Regulates Expression of Genes Involved in Cell Cycle Progression of Metastatic Models of Prostate Cancer.

The effects of sarcosine on the processes driving prostate cancer (PCa) development remain still unclear. Herein, we show that a supplementation of metastatic PCa cells (androgen independent PC-3 and androgen dependent LNCaP) with sarcosine stimulates cells proliferation in vitro. Similar stimulatory effects were observed also in PCa murine xenografts, in which sarcosine treatment induced a tumor growth and significantly reduced weight of treated mice (p < 0.05). Determination of sarcosine metabolism-related amino acids and enzymes within tumor mass revealed significantly increased glycine, serine and sarcosine concentrations after treatment accompanied with the increased amount of sarcosine dehydrogenase. In both tumor types, dimethylglycine and glycine-N-methyltransferase were affected slightly, only. To identify the effects of sarcosine treatment on the expression of genes involved in any aspect of cancer development, we further investigated expression profiles of excised tumors using cDNA electrochemical microarray followed by validation using the semi-quantitative PCR. We found 25 differentially expressed genes in PC-3, 32 in LNCaP tumors and 18 overlapping genes. Bioinformatical processing revealed strong sarcosine-related induction of genes involved particularly in a cell cycle progression. Our exploratory study demonstrates that sarcosine stimulates PCa metastatic cells irrespectively of androgen dependence. Overall, the obtained data provides valuable information towards understanding the role of sarcosine in PCa progression and adds another piece of puzzle into a picture of sarcosine oncometabolic potential.

Distinct Osteomimetic Response of Androgen-Dependent and Independent Human Prostate Cancer Cells to Mechanical Action of Fluid Flow: Prometastatic Implications.

Prostate cancer frequently expresses an osteomimetic phenotype, but it is unclear how it is regulated and what biological and clinical implications it confers. Because mechanical forces physiologically regulate bone-remodeling activity in osteocytes, we hypothesized that mechanical action of fluid flow (MAFF) at the cancer microenvironment may similarly foster prostate cancer cell osteomimicry. We showed that in vitro MAFF on androgen-dependent (LNCap) and androgen-independent (PC3) prostate cancer cells remarkably increased OPG, VEGF, RunX2, PTH1R, and PTHrP gene expression in both cell lines irrespective of their androgen dependency. MAFF also altered the cytokine secretion pattern of prostate cancer cells, including Ang2, SCF, and TNFα increase with TRAIL decrease in the supernatant of both cell lines; preferential increase of Leptin and PDGF-BB in LnCap and of VEGF, IL-8, and G-CSF in PC3; and exclusive increase of FGFβ, MIF, and PECAM-1 with HGF decrease in LnCap, and of TGBβ1, HGF, M-CSF, CXCL1, and CCL7 with NGF decrease in PC3. Murine MLO-Y4 osteocyte-conditioned medium (CM) abrogated M-CSF, G-CSG, IL-8, TNFα, and FGFβ secretion-stimulating activity of mechanical stimulation on PC3 cells, and did the opposite effect on LnCap cells. However, MAFF fostered osteomimetic gene expression response of PC3 cells, but not of LnCap cells, to mechanically stimulated osteocyte-CM. Moreover, it abrogated TNFα and IL-8 secretion inhibitory effect of osteocyte-CM on mechanically stimulated PC3 cells and G-CSF, TNFα, and FGFβ-stimulating effect on mechanically stimulated LnCap cells. MAFF activated osteoblast-like phenotype of prostate cancer cells and altered their responses to osteocyte soluble factors. It also induced osteocyte production of osteomimetic gene expression- and cytokine secretion-stimulating factors for prostate cancer cells, particularly, when they were mechanically stimulated. Importantly, MAFF induced a prometastatic response in androgen-independent prostate cancer cells, suggesting the interest of mechanical stimulation-dependent transcription and secretion patterns as diagnostic biomarkers, and as therapeutic targets for the screening of bone-metastasizing phenotype inhibitors upregulated during prostate cancer cell response to MAFF at the cancer microenvironment. Prostate 77:321-333, 2017. © 2016 Wiley Periodicals, Inc.

Abstract C41: Altered control of intracellular androgen availability by glucuronidation contributes to loss of androgen dependence in prostate tumor cells

Abstract Locally advanced and metastatic cancers are frequently treated by androgen deprivation therapy. Tumors that recur despite this treatment, termed castration resistant prostate cancer, are highly aggressive and have acquired the capacity to survive and expand in conditions of low circulating androgens. Prostate epithelial cells control the potency and overall availability of androgen hormones in part by inactivation and excretion of excess quantities. The enzymes that catalyze inactivation, specifically by covalently modifying the hormone with a glucuronate adduct, are called UDP-glucuronosyltransferases (UGT). Genetic mutations that reduce levels of the UGT isozymes needed to eliminate excess androgens from the prostate have been correlated with prostate cancer incidence. The ability to alter androgen elimination at the level of individual cells within a tumor is one mechanism by which tumors become heterogeneous with respect to molecular features and therapeutic resistance. UDP-glucose dehydrogenase (UGDH) catalyzes the NAD+-dependent oxidation of UDP-glucose to UDP-glucuronate, an essential precursor for androgen inactivation by the prostate glucuronidation enzymes UGT2B15 and UGT2B17. Our laboratory has found that non-metastatic prostate tumor cell lines express UGDH in an androgen sensitive manner, that its expression increases the level of UDP-glucuronate, and that these precursors are then channeled to the UGT-catalyzed glucuronidation pathway. The level of UGDH is androgen controlled and serves to regulate tumor cell growth, inversely to its expression. Loss of androgen stimulated UGDH, or pharmacological antagonism of its activity, translated to increased proliferation rate, specifically of androgen dependent tumor cells. We knocked down expression of UGDH in androgen dependent cells to test the role of UGDH in driving cellular glucuronate levels, intratumoral steroid availability, and response to androgen. Flux of UDP sugar metabolites and levels of androgen glucuronide accumulation were compared in control and UGDH knockdown lines by liquid chromatography and mass spectrometry analysis. Effects on androgen sensitive gene expression were monitored by western analysis. Selection of manipulated cells in decremental DHT levels that simulated androgen deprivation was found to reduce steady state UGDH expression, thereby activating the UGT-driven steroid clearance mechanism, as reflected in increased PSA levels and de-repression of UGT2B17. Whereas restimulation of control transfectants with physiological levels of DHT could only increase UGDH expression and maintain full androgen sensitivity in cells experiencing complete androgen loss, the knockdown of UGDH permitted cells to accumulate UDP-glucuronate pools, and decreased UGT-driven glucuronidation of DHT and androsterone. These results support a model in which maintenance of partial UGDH activity through gradual androgen deprivation facilitates loss of sensitivity in androgen elimination pathways and suggests adjuvant UGDH inhibition could prevent or delay onset of castration resistant recurrence. Citation Format: Michelle Palmer, Linlin Ma, Trevor Romsdahl, Qin Wei, Eileen Loughman, Jonathan Markham, Joseph Barycki, Melanie A. Simpson. Altered control of intracellular androgen availability by glucuronidation contributes to loss of androgen dependence in prostate tumor cells. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C41.

Abstract 1056: Inhibition of hexose monophosphate pathway promotes castration resistant prostate cancer

Abstract Prostate Cancer (PCa) is the second highest cause of cancer-related death in men in the US. PCa is androgen dependent when organ-confined and is conventionally treated using surgery or using a combination of anti-androgens and radiation therapy. However, in about 30% of the patients tumor recurs and are initially administered androgen deprivation therapy (ADT). Majority of the patients become resistant to ADT and develop hormone-refractory disease also termed castration-resistant prostate cancer (CRPC), which is lethal. Currently, the molecular and biochemical alterations driving CRPC are not well understood. Using a novel network-based integrative approach, we show distinct alterations in the Hexosamine Biosynthetic Pathway (HBP) to be critical for sustaining the castrate resistant state. Our data suggests expression of key HBP enzymes to be significantly elevated in androgen dependent (AD) PCa while interestingly enough, relatively diminished in CRPC. Genetic loss of function experiments for these HBP enzymes in CRPC-like cells had tumor promoting effect both in vitro and in vivo. This was mediated by alterations in either PI3K-AKT pathway or SP1-ChREBP (SP1- carbohydrate response element binding protein) network in CRPC cells containing full length androgen receptor (AR) or its splice variant AR-V7, respectively. Strikingly, addition of HBP metabolite UDP-N-acetylglucosamine (UDP-GlcNAc) or glucosamine (GlcN) to CRPC-like cells attenuated tumor cell proliferation, both in vitro and in animal studies. Interestingly, these metabolites demonstrated additive efficacy when combined with enzalutamide in vitro. These findings are particularly significant given that the CRPC-like cells tested, inclusive of those containing AR-V7 variant, are inherently resistant to enzalutamide. These observations demonstrate the therapeutic value of targeting altered HBP in CRPC. Citation Format: Akash K. Kaushik, Ali Shojaie, Katrin Panzitt, Rajni Sonavane, Harene Venghatakrishnan, Mohan Manikkam, Alexander Zaslavsky, Vasanta Putluri, Vihas Vasu, Yiqing Zhang, Ayesha Khan, Stacy Lloyd, Adam Szafran, Subhamoy Dasgupta, David Bader, Fabio Stossi, Hangwen Li, Susmita Samanta, Xuhong Cao, Efrosini Tsouko, Shixia Huang, Daniel Frigo, Lawrence Chan, Dean Edwards, Benny Kaipparettu, Nicholas Mitsiades, Nancy Weigel, Michael Mancini, Michael Ittmann, Arul Chinnaiyan, Nagireddy Putluri, Ganesh Palapattu, George Michailidis, Arun Sreekumar. Inhibition of hexose monophosphate pathway promotes castration resistant prostate cancer. [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 1056.

Abstract 2990: Sulforaphane enhances the anti-cancer efficacy of anti-androgens in prostate cancer cells (LNCaP and C4-2B) by increasing androgen receptor (AR) degradation

Abstract Introduction: Prostate Cancer (PC) cells utilize androgen for their growth. Therefore, androgen deprivation therapy (ADT) that blocks both systemic androgen production and androgen receptor (AR) signaling remains the mainstay of PC treatment. However, despite the efficacy of AR antagonists such as bicalutamide (BIC) and enzalutamide (ENZ), their sub-therapeutic efficacy in tumor microenvironments enable the selection and outgrowth of castration resistant PC (CRPC). Interestingly, CRPC cells continue to express AR and exploit the intratumoral androgen. Hence, alternate strategies to suppress AR expression in PC cells will be needed to enhance the efficacy of BIC and ENZ. The phytochemical, sulforaphane (SFN) is known to decrease AR protein levels. We hypothesize that co-exposure to SFN will increase the anti-cancer efficacy of BIC/ENZ. Methods: The AR expressing PC lines, both androgen-dependent (AD) LNCaP cells and androgen-independent CRPC cells (C4-2B), were used to investigate the effects of BIC or ENZ, alone and in combination with SFN. The following cellular parameters were evaluated: (a) cell proliferation by MTT-assay; (b) clonogenic ability by colony forming unit (CFU); (c) AR expression by immunoblot analysis; (d) subcellular AR localization by immunofluorescence microscropy (IFM); (e) AR and prostate specific antigen (PSA) gene expression by qRT-PCR; and (e) PSA protein secretion by ELISA. Studies were carried out under hormone deprived (charcoal-stripped FBS) conditions and/or in presence of AR agonist, R1881. Results: Co-exposure to SFN (10μM) significantly (p&amp;lt;0.05) enhanced the anti-proliferative effects of BIC (50μM) or ENZ (20μM) at 48 h post treatment, and suppressed R1881 stimulated PC growth, as well. At much lower concentrations, SFN (0.2μM) co-exposure enhanced the suppressive effects of BIC (0.5μM) or ENZ (0.2μM) on both the number and size of CFUs at 14 days. Western blot analysis revealed that SFN decreases AR protein levels in a time- and dose-dependent manner. IFM analysis indicated that SFN suppresses R1881-stimulated AR nuclear localization. Cycloheximide treatment suggested that SFN's effect on AR is primarily regulated at the translational level. Experiments with MG-132, a proteasomal inhibitor indicated that the SFN-induced AR degradation is partially regulated by the proteasomal pathway. Studies with qRT-PCR showed that SFN enhances the efficacy of BIC in suppressing PSA mRNA levels and decreased PSA protein secretion was also corroborated by ELISA. Conclusion: Sulforaphane decreases AR levels in both LNCaP and C4-2B cells to increase the anti-cancer efficacy of AR antagonists in a synergistic manner. This implies that adjuvant therapy with this safe phytochemical may be very promising. Citation Format: Namrata Khurana, Sudha Talwar, Partha Chandra, Asim B. Abdel-Mageed, Debasis Mondal, Suresh Sikka. Sulforaphane enhances the anti-cancer efficacy of anti-androgens in prostate cancer cells (LNCaP and C4-2B) by increasing androgen receptor (AR) degradation. [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 2990.

Abstract 1110: Inhibition of histone deacetylase induces miR-320-mediated androgen receptor suppression in prostate cancer

Abstract Targeting androgen receptor (AR) by pharmacological intervention is one of the effective approaches for treatment of malignant prostate cancers. Histone deacetylase (HDAC) alters epigenetic status of tumor-associated genes, including microRNAs (miRNAs), and affects the behavior of cancers. Here we examined molecular effects of a HDAC inhibitor, OBP-801, on AR expression and tumor cell growth in prostate cancers. Treatment of OBP-801 efficiently suppressed cell growth of three prostate cancer lines (22Rv1, VCaP, and LNCaP), together with AR downregulation, regardless of their hormone sensitivity. Intriguingly, this effect by OBP-801 is not due to downregulation of transcriptional activity of the AR gene, but due to post-transcriptional regulation, namely miRNA-mediated suppression. Among the upregulated miRNAs after OBP-801 treatment in three prostate cancer cell lines, miR-320a, of which expression was significantly correlated with prognosis of prostate cancers (P = 0.0185), was the most closely associated with AR expression. miR-320a mimic suppressed AR protein expression together with growth suppression, while anti-miR320a significantly abrogated the growth suppression by OBP-801 treatment. Fluorescent in situ hybridization analysis revealed that miR-320a was highly expressed in human normal prostate luminal cells but rarely expressed in prostate cancer cells. In AR-dependent prostate tumorigenic rat model, OBP-801 treatment profoundly increased the miR-320 expression and repressed prostate tumorigenesis. Our data demonstrated that OBP-801 effectively suppressed AR activity via epigenetic upregulation of miR-320a, which resulted in tumor cell growth suppression of prostate cancers. OBP-801 may be a potent drug as an AR targeting therapy in AR positive prostate cancer regardless of androgen dependency. Citation Format: Shinya Sato, Keisuke Katsushima, Keiko Shinjo, Satoru Takahashi, Yutaka Kondo. Inhibition of histone deacetylase induces miR-320-mediated androgen receptor suppression in prostate cancer. [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 1110.

Histone Deacetylase Inhibition in Prostate Cancer Triggers miR-320-Mediated Suppression of the Androgen Receptor.

Targeting androgen receptor (AR) by pharmacologic intervention is one of the effective approaches for treatment of malignant prostate cancers. Histone deacetylase (HDAC) alters the epigenetic status of tumor-associated genes, including those for miRNAs (miRNA), and affects the behavior of cancers. Here, we examined the molecular effects of a HDAC inhibitor, OBP-801, on AR expression and tumor cell growth in prostate cancers. Treatment with OBP-801 efficiently suppressed cell growth of three prostate cancer lines (22Rv1, VCaP, and LNCaP), together with AR downregulation, regardless of their hormone sensitivity. Intriguingly, this effect by OBP-801 was not due to decreased transcriptional activity of the AR gene, but due to posttranscriptional regulation, namely by miRNA-mediated suppression. Among the upregulated miRNAs after OBP-801 treatment in the three prostate cancer cell lines, miR-320a, whose expression was significantly correlated with prognosis of prostate cancers (P = 0.0185), was the most closely associated with AR expression. An miR-320a mimic suppressed AR protein expression together with growth suppression, while anti-miR-320a oligonucleotide significantly abrogated the growth suppression by OBP-801 treatment. FISH analysis revealed that miR-320a was highly expressed in human normal prostate luminal cells, but was rarely expressed in prostate cancer cells. In an AR-dependent prostate tumorigenic rat model, OBP-801 treatment profoundly increased miR-320a expression and repressed prostate tumorigenesis. Our data demonstrated that OBP-801 effectively suppressed AR activity via epigenetic upregulation of miR-320a, which resulted in tumor cell growth suppression of prostate cancers. OBP-801 may be a potent AR-targeting therapeutic reagent in AR-positive prostate cancer regardless of androgen dependency. Cancer Res; 76(14); 4192-204. ©2016 AACR.

Loss of exogenous androgen dependence by prostate tumor cells is associated with elevated glucuronidation potential.

Prostate epithelial cells control the potency and availability of androgen hormones in part by inactivation and elimination. UDP-glucose dehydrogenase (UGDH) catalyzes the NAD(+)-dependent oxidation of UDP-glucose to UDP-glucuronate, an essential precursor for androgen inactivation by the prostate glucuronidation enzymes UGT2B15 and UGT2B17. UGDH expression is androgen stimulated, which increases the production of UDP-glucuronate and fuels UGT-catalyzed glucuronidation. In this study, we compared the glucuronidation potential and its impact on androgen-mediated gene expression in an isogenic LNCaP model for androgen-dependent versus castration-resistant prostate cancer. Despite significantly lower androgen-glucuronide output, LNCaP 81 castration-resistant tumor cells expressed higher levels of UGDH, UGT2B15, and UGT2B17. However, the magnitude of androgen-activated UGDH and prostate-specific antigen (PSA) expression, as well as the androgen receptor (AR)-dependent repression of UGT2B15 and UGT2B17, was blunted several-fold in these cells. Consistent with these results, the ligand-activated binding of AR to the PSA promoter and subsequent transcriptional activation were also significantly reduced in castration-resistant cells. Analysis of the UDP-sugar pools and flux through pathways downstream of UDP-glucuronate production revealed that these glucuronidation precursor metabolites were channeled through proteoglycan and glycosaminoglycan biosynthetic pathways, leading to increased surface expression of Notch1. Knockdown of UGDH diminished Notch1 and increased glucuronide output. Overall, these results support a model in which the aberrant partitioning of UDP-glucuronate and other UDP-sugars into alternative pathways during androgen deprivation contributes to the loss of prostate tumor cell androgen sensitivity by promoting altered cell surface proteoglycan expression.

Cytoplasmic, full length and novel cleaved variant, TBLR1 reduces apoptosis in prostate cancer under androgen deprivation.

TBLR1/TBL1XR1, a core component of the nuclear receptor corepressor (NCoR) complex critical for the regulation of multiple nuclear receptors, is a transcriptional coactivator of androgen receptor (AR) and functions as a tumor suppressor when expressed in the nucleus in prostate. Subcellular localization of a protein is critical for its function, and although TBLR1, as a transcriptional cofactor, has been primarily viewed as a nuclear protein, many cells also express variable levels of cytoplasmic TBLR1 and its cytoplasmic specific functions have not been studied. Prostate cancer (PCa) cells express moderately higher level of cytoplasmic TBLR1 compared to benign prostate cells. When comparing androgen-dependent (AD) to androgen-independent (AI) PCa, AI cells contain very high levels of TBLR1 cytoplasmic expression and low levels of nuclear expression. Overexpression of cytoplasmic TBLR1 in AD cells inhibits apoptosis induced by androgen deprivation therapy, either in an androgen free condition or in the presence of bicalutamide. Additionally, we identified a cytoplasmic specific isoform of TBLR1 (cvTBLR1) approximately 5 kDa lower in molecular weight, that is expressed at higher levels in AI PCa cells. By immunoprecipitation, we purified cvTBLR1 and using mass spectrometry analysis combined with N-terminal TMPP labeling and Edman degradation, we identified the cleavage site of cvTBLR1 at amino acid 89, truncating the first 88 amino acids of the N-terminus of the full length protein. Functionally, cvTBLR1 expressed in the cytoplasm reduced apoptosis in PCa cells and promoted growth, migration, and invasion. Finally, we identified a nuclear export signal sequence for TBLR1 cellular localization by deletion and site-directed mutagenesis. The roles of TBLR1 and cvTBLR1 provide novel insights into the mechanism of castration resistance and new strategies for PCa therapy.

Abstract A11: Downregulation of NRH:quinone oxidoreductase 2(NQO2) in the LNCaP prostate cancer progression model system: Implications to metastasis

Abstract Prostate cancer is characterized by an innate capacity to produce higher concentrations of reactive oxygen species (ROS). ROS is also a hallmark for highly aggressive disease. Our laboratory has focused on the molecular mechanisms of inhibition of prostate cancer bone metastasis by the naturally occurring plant based anti-oxidant compound curcumin, arising from the rhizomes of Curcuma Longa. These studies highlight the importance of studying the mechanisms of redox homeostasis in normal cells and how they are subverted in cancer cells. The LNCaP prostate cancer progression model system as exemplified by the development of C4, C4-2 and C4-2B ( bone metastatic) sublines derived from the parental LNCaP prostate cancer cells offer an unique avenue to study the role of redox systems in the progression to metastatic disease, culminating in the establishment of osseous metastases. The mechanisms of ROS production and destruction, the cellular machinery and the enzyme systems involved, the relationship of ROS to cellular oncogene activation and the antagonistic duality of oncogenes and tumor suppressor genes with respect to ROS homeostasis have been subjects of intense investigation. How the prostate cancer cells metabolize ROS to achieve their proliferative and metastatic potential is not completely understood. We chose to study one redox system orchestrated by the expression of NADPH:Quinone Oxidoreductase1 (NQO1) and the Nicotinamide Riboside (NRH): Quinone Oxidoreductase 2 (NQO2) in the LNCaP progression model system. Particularly, the enzyme NQO2 has been shown earlier to be the target of another polyphenolic chemopreventive antioxidant resveratrol. The role of NQO2 as a phase II detoxification enzyme acting on several xenobiotics is well characterized. However, the role of NQO2 in regulating ROS and hence cancer progression is only beginning to be understood. With this background, we investigated the potential role of NQO2/NQO1 expression in the transition from androgen dependence to androgen independence and to acquiring bone metastatic potential in C4-2B prostate cancer cells. Our studies indicate that 1) there is a down regulation of NQO2 as the androgen dependent LNCaP cells progress towards androgen independence in C4 and C4-2 cells. In C4-2B cells, there is a barely detectable level of NQO2. In contrast, there is very little change in the level of expression of NQO1; 2) Characteristically, the intracellular ROS production progressively increases from LNCaP to C4-2B; 3) In parallel, there is an up regulation of caveolin-1 as the LNCaP cells progress to bone metastatic C4-2B and 4) a small fraction of NQO2 could be detected in the detergent resistant membrane (DRM) fraction consisting of lipid rafts and caveolae. Our studies suggest that caveolin-1 may play a role in the membrane sequestration of the largely cytoplasmic NQO2 in LNCaP cells and that this interaction could be lost in bone metastatic C4-2B cells. The significance of these findings in relation to the metastatic potential of C4-2B cells will be discussed. Citation Format: Ankeeta Shah, Tze-Chen Hsieh, Rajamma Mathew, Joseph M. Wu, Thambi Dorai. Downregulation of NRH:quinone oxidoreductase 2(NQO2) in the LNCaP prostate cancer progression model system: Implications to metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A11.

Fatty acid activated PPARγ promotes tumorigenicity of prostate cancer cells by up regulating VEGF via PPAR responsive elements of the promoter.

In previous work, it is suggested that the excessive amount of fatty acids transported by FABP5 may facilitate the malignant progression of prostate cancer cells through a FABP5-PPARγ-VEGF signal transduction axis to increase angiogenesis. To further functionally characterise the FABP5-PPARγ-VEGF signal transduction pathway, we have, in this work, investigated the molecular mechanisms involved in its tumorigenicity promoting role in prostate cancer. Suppression of PPARγ in highly malignant prostate cancer cells produced a significant reduction (up to 53%) in their proliferation rate, invasiveness (up to 89%) and anchorage-independent growth (up to 94%) in vitro. Knockdown of PPARγ gene in PC3-M cells by siRNA significantly reduced the average size of tumours formed in nude mice by 99% and tumour incidence by 90%, and significantly prolonged the latent period by 3.5 fold. Results in this study combined with some previous results suggested that FABP5 promoted VEGF expression and angiogenesis through PPARγ which was activated by fatty acids transported by FABP5. Further investigations showed that PPARγ up-regulated VEGF expression through acting with the PPAR-responsive elements in the promoter region of VEGF gene in prostate cancer cells. Although androgen can modulate VEGF expression through Sp1/Sp3 binding site on VEGF promoter in androgen-dependent prostate cancer cells, this route, disappeared as the cells gradually lost their androgen dependency; was replaced by the FABP5-PPARγ-VEGF signalling pathway. These results suggested that the FABP5-PPARγ-VEGF signal transduction axis, rather than androgen modulated route, may be a more important novel therapeutic target for angiogenesis-suppression treatment of castration resistant prostate cancer.

Effects of a novel carbocyclic analog of pyrrolo[2,3-d]pyrimidine nucleoside on pleiotropic induction of cell death in prostate cancer cells with different androgen responsiveness.

Prostate cancer is the most frequently diagnosed cancer and is one of the leading causes of male cancer death in the world. Recently, in the course of our screening for a novel anticancer compound, we synthesized carbocyclic analogs of pyrrolo[2,3-d]pyrimidine nucleoside; compounds 5, and 6. In the current study, we report the effects of compound 5 on pleiotropic induction of cell death via up-regulation of AR-associated p21(Cip1) protein in prostate cancer cells with different androgen responsiveness, such as LNCaP (androgen-dependent and -sensitive), LNCaP(C4-2) (androgen-independent and -sensitive; androgen-refractory), and DU145 (androgen-independent and -insensitive) cells. The treatment of LNCaP cells with 6 μM compound 5 for 24 h stimulated the androgen receptor (AR) activity and dramatically up-regulated transcription (56-fold) of p21(Cip1), which, in turn, induces typical apoptosis in the cells. However, induction of apoptosis through up-regulation (23-fold) of AR-associated p21(Cip1) achieved in LNCaP(C4-2) cells was possible by intensive cell treatment with compound 5 (9 μM, 48 h), because the cells are less sensitive and independent to androgen than LNCaP cells. Furthermore, 6 μM compound 5-treated DU145 cells, which exhibit extremely low AR activation due to no androgen responsiveness and dependency, showed neither up-regulation of p21(Cip1) nor apoptotic induction. Instead, a different type of cell death, autophagy-like death through the LC3B-associated autophagosome formation, was obviously induced in DU145 cells. Taken together, our results suggest that pleiotropic induction of prostate cancer cell death by compound 5 is determined by how efficiently and how abundantly androgen-dependent activation of the AR occurs, whereas compound 6 shows no induction of apoptosis in LNCaP cells.

Abstract A03: Procyanidin B2 3,3”-di-O-gallate from grape seed causes metabolic alterations in prostate cancer cells: 1H-, 13C- and 31P-NMRS-based metabolomics study

Abstract Herein, we assessed the efficacy of procyanidins from grape seed against a panel of human prostate cancer (PCa) cell lines which ranged from ‘classical’ cell lines to the new variants that differed in their androgen responsiveness, castration resistance, and metastatic potential. The classical cell lines chosen were PC3, DU145 and LNCaP. Of these cell lines: PC3 and DU145 do not need androgens for growth, i.e, these are androgen independent (AI); they also lack androgen receptors (AR). The LNCaP cell line on the other hand, demonstrates androgen sensitivity (AS) and also requires androgens for its growth; while it harbors a mutated AR. Of the new variants of PCa cell lines available, we further chose a castration resistant variant of LNCaP, C4-2B, derived from the xenografts of castration resistant LNCaP subline-C4 in castrated mice. C4-2B thus represents castration resistant PCa (CRPC) cell line; while it does not require androgen for growth it demonstrates androgen sensitivity due to the presence of AR and is thus categorized as CRPC (AI/ AS) variant. Another PCa cell line, 22Rv1, which was chosen, does not require androgens for growth but is androgen responsive and like C4-2B is categorized as CRPC (AI/ AS).Trypan Blue dye exclusion assays utilizing total procyanidins from grape seed or its active constituent procyanidin B2 3,3”-di-O-gallate (B2G2) were performed to determine procyanidin effects, as a function of time, on the viability of the selected human PCa cell lines. Results indicated that the androgen dependent LNCaP cell line and its castration resistant variant, C4-2B, were more sensitive to procyanidins treatment at lower doses; importantly, another castration resistant cell line, 22Rv1, also showed significant decrease in cell viability at lower doses. While at these lower doses significant cell death was observed even at time points as early as 12 h in these cell lines; the classical AI cell lines PC3 and DU145 were most resistant to procyanidin treatment at lower doses and showed sensitivity (growth inhibition and cell death) only at higher doses after 72 h of procyanidins exposure. On similar lines, cancer stem cells (CD44+alpha2beta1high cells) isolated from PC3 and DU145 cells were also more resistant to treatment with procyanidins in prostasphere formation assays compared to other PCa cell lines. To understand the differential efficacy of procyanidins or its active constituent B2G2 towards different PCa cell lines, we carried out a metabolomics study utilizing quantitative high-resolution nuclear magnetic resonance spectroscopy (1H-, 13C- and 31P-NMR) to assess the metabolic profile and energy state of the B2G2 treated PCa cells. We examined global metabolic profile, including glucose metabolism, energy state, and lipid metabolism in these cells after B2G2 treatment. The time-course of B2G2 effects on glucose uptake and lactate release was also studied in the media of B2G2-treated PCa cells from 4-72 hrs of treatment. Results indicated that there was differential effect of B2G2 on mitochondrial glucose metabolism in these cells. While glucose uptake was markedly reduced as a function of time, there was also a significant decrease in extracellular lactate export with increased B2G2 exposure-time. Importantly, B2G2 preserved citrate concentration in the PCa cells as indicated by increase in citrate levels after treatments; citrate generation is indicative of normal secretion functions of the prostate epithelial cells. The fact that the citrate was not further utilized for cholesterol synthesis was also confirmed by decrease in cholesterol levels in these cells, indicating decreased cholesterogenesis by B2G2. Taken together these studies show that the procyanidins from grape seed differentially induce metabolic alterations in various PCa cell lines; these modulatory effects can be correlated with the effects on cell growth, proliferation and death, as well as the androgen dependency/ or lack of it. Citation Format: Komal Raina, Dileep Kumar, Rajesh Agarwal, Chapla Agarwal. Procyanidin B2 3,3”-di-O-gallate from grape seed causes metabolic alterations in prostate cancer cells: 1H-, 13C- and 31P-NMRS-based metabolomics study. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A03.

Mitochondrial oncobioenergetic index: A potential biomarker to predict progression from indolent to aggressive prostate cancer.

Mitochondrial function is influenced by alterations in oncogenes and tumor suppressor genes and changes in the microenvironment occurring during tumorigenesis. Therefore, we hypothesized that mitochondrial function will be stably and dynamically altered at each stage of the prostate tumor development. We tested this hypothesis in RWPE-1 cells and its tumorigenic clones with progressive malignant characteristics (RWPE-1 < WPE-NA22 < WPE-NB14 < WPE-NB11 < WPE-NB26) using high-throughput respirometry. Our studies demonstrate that mitochondrial content do not change with increasing malignancy. In premalignant cells (WPE-NA22 and WPE-NB14), OXPHOS is elevated in presence of glucose or glutamine alone or in combination compared to RWPE-1 cells and decreases with increasing malignancy. Glutamine maintained higher OXPHOS than glucose and suggests that it may be an important substrate for the growth and proliferation of prostate epithelial cells. Glycolysis significantly increases with malignancy and follow a classical Warburg phenomenon. Fatty acid oxidation (FAO) is significantly lower in tumorigenic clones and invasive WPE-NB26 does not utilize FAO at all. In this paper, we introduce for the first time the mitochondrial oncobioenergetic index (MOBI), a mathematical representation of oncobioenergetic profile of a cancer cell, which increases significantly upon transformation into localized premalignant form and rapidly falls below the normal as they become aggressive in prostate tumorigenesis. We have validated this in five prostate cancer cell lines and MOBI appears to be not related to androgen dependence or mitochondrial content, but rather dependent on the stage of the cancer. Altogether, we propose that MOBI could be a potential biomarker to distinguish aggressive cancer from that of indolent disease.

Abstract A28: N-Myc drives small cell neuroendocrine cancer initiated from human prostate basal cells

Abstract Human small cell prostate carcinoma (SCPC) represents up to 25% of lethal castration-resistant prostate cancer. Molecular characterization of a panel of SCPCs recently identified frequent genomic amplification of both MYCN and AURKA. The oncogenic function of N-Myc has been characterized in many cancer types but its role in the initiation of human SCPC has not been defined. To investigate the function of N-Myc in human prostate transformation, we utilized a tissue regeneration model of human prostate cancer. Human prostate basal epithelial cells were isolated from benign primary prostate tissue from four independent human donors, infected with lentiviruses expressing the oncogenes N-Myc and activated (myristoylated) AKT1, mixed with mouse urogenital sinus mesenchyme, and grafted subcutaneously in immunodeficient NOD-SCID-IL2Rγnull mice. Palpable tumors were appreciated within 2-3 months and histological evaluation revealed regions of mixed high-grade adenocarcinoma and small cell carcinoma. The small cell carcinoma histology showed typical findings of high nuclear-to-cytoplasmic ratio, nuclear molding, stippled chromatin, and frequent mitotic and apoptotic features. Immunohistochemical analysis demonstrated low to absent androgen receptor (AR) staining in both the adenocarcinoma and small cell carcinoma. Further, we found heterogeneous staining for neuroendocrine markers including neural cell adhesion molecule 1, chromogranin A, synaptophysin, thyroid transcription factor 1, and forkhead box A2 in the regions of small cell carcinoma in support of the histological diagnosis. To assess the androgen dependence of tumor growth, regenerated N-Myc and activated AKT1 tumors derived from the lentiviral infection of primary human prostate basal cells were passaged in either intact or castrate immunodeficient mice. Tumors were propagated both in the presence and absence of androgens and showed a small but progressive enrichment of the small cell carcinoma over the adenocarcinoma with successive passage in castrate hosts. Molecular profiling by RNA-Seq of three regenerated N-Myc and activated AKT1 tumors showed a significant resemblance to a subset of human SCPC clinical specimens. Further, the adenocarcinoma and small cell carcinoma components of each tumor were remarkably similar at the transcriptional level despite their differences in histological appearance. In conclusion, we have established a novel model of human small cell prostate cancer arising from benign human prostate basal cells. Overexpression of N-Myc and activated AKT1 in basal cells produces tumors that recapitulate the histology, immunophenotype, and transcriptional profile of human SCPC. These findings support a direct role for N-Myc in the neuroendocrine transformation of basal cells in the human prostate. Further studies are ongoing to elucidate the mechanisms by which N-Myc induces a neuroendocrine differentiation program in prostate cancer. Citation Format: John K. Lee, Bryan A. Smith, John W. Phillips, Jungwook Park, Tanya I. Stoyanova, Robert Baertsch, Artem Sokolov, Colleen Mathis, Donghui Cheng, Joshua M. Stuart, Jiaoti Huang, Owen N. Witte. N-Myc drives small cell neuroendocrine cancer initiated from human prostate basal cells. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr A28.

The lncRNA DRAIC/PCAT29 Locus Constitutes a Tumor-Suppressive Nexus.

Long noncoding RNAs (lncRNA) are emerging as major regulators of cellular phenotypes and implicated as oncogenes or tumor suppressors. Here, we report a novel tumor-suppressive locus on human chromosome 15q23 that contains two multiexonic lncRNA genes of 100 kb each: DRAIC (LOC145837) and the recently reported PCAT29. The DRAIC lncRNA was identified from RNA-seq data and is downregulated as prostate cancer cells progress from an androgen-dependent (AD) to a castration-resistant (CR) state. Prostate cancers persisting in patients after androgen deprivation therapy (ADT) select for decreased DRAIC expression, and higher levels of DRAIC in prostate cancer are associated with longer disease-free survival (DFS). Androgen induced androgen receptor (AR) binding to the DRAIC locus and repressed DRAIC expression. In contrast, FOXA1 and NKX3-1 are recruited to the DRAIC locus to induce DRAIC, and FOXA1 specifically counters the repression of DRAIC by AR. The decrease of FOXA1 and NKX3-1, and aberrant activation of AR, thus accounts for the decrease of DRAIC during prostate cancer progression to the CR state. Consistent with DRAIC being a good prognostic marker, DRAIC prevents the transformation of cuboidal epithelial cells to fibroblast-like morphology and prevents cellular migration and invasion. A second tumor-suppressive lncRNA PCAT29, located 20 kb downstream of DRAIC, is regulated identically by AR and FOXA1 and also suppresses cellular migration and metastasis. Finally, based on TCGA analysis, DRAIC expression predicts good prognosis in a wide range of malignancies, including bladder cancer, low-grade gliomas, lung adenocarcinoma, stomach adenocarcinoma, renal clear cell carcinoma, hepatocellular carcinoma, skin melanoma, and stomach adenocarcinoma. This study reveals a novel tumor-suppressive locus encoding two hormone-regulated lncRNAs, DRAIC and PCAT29, that are prognostic for a wide variety of cancer types.

Increased acid ceramidase expression depends on upregulation of androgen-dependent deubiquitinases, USP2, in a human prostate cancer cell line, LNCaP

Acid ceramidase (ACDase) metabolizes ceramide to sphingosine, leading to sphingosine 1-phosphate production. Reportedly, ACDase has been upregulated in prostate cancer. However, its regulatory mechanism remains unclear. LNCaP (androgen-sensitive prostate cancer cell line) but not PC3 and DU-145, (androgen-unresponsive cell lines) exhibited the highest ACDase protein. Among three cell lines, ASAH1 mRNA level was not correlated with ACDase protein expression, and the 5'-promoter activity did not show androgen dependency, suggesting the post-transcriptional regulation of ACDase in LNCaP cells. Based on these results, LNCaP was analysed further. Casodex, androgen receptor antagonist, and charcoal-stripped FCS (CS-FCS) decreased ACDase protein and activity, whereas dihydrotestosterone in CS-FCS culture increased ACDase protein and enzyme activity. MG132, a proteasome inhibitor, prevented the decrease of ACDase protein when cultured in CS-FCS, suggesting the involvement of ubiquitin/proteasome system. Reportedly, USP2, a deubiquitinase, plays an important role in LNCaP cells. USP2 siRNA decreased ACDase protein, whereas USP2 overexpression increased ACDase protein of LNCaP cells. However, SKP2, an ubiquitin E3 ligase known to be active in prostate cancer, did not affect androgen-dependent ACDase expression in LNCaP cells. Thus, ACDase regulation by androgen in androgen-sensitive LNCaP cells is mainly due to its prolonged protein half-life by androgen-stimulated USP2 expression.

Neurones in the preoptic area of the male goldfish are activated by a sex pheromone 17α,20β-dihydroxy-4-pregnen-3-one.

Pheromones are interesting molecules given their ability to evoke changes in the endocrine state and behaviours of animals. In goldfish, a sex pheromone, 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P), which is released by preovulatory females, is known to trigger the elevation of luteinising hormone (LH) levels, as well as reproductive behaviour in males. Interestingly, when 11-ketotestosterone (11-KT) is implanted into adult female fish, LH levels increase in response to the pheromone at any time of the day, which is normally a male-specific response. However, the neural mechanisms underlying the male-specific information processing of 17,20β-P and its androgen dependence are yet unknown. In the present study, we focused on the preoptic area (POA), which plays important roles in the regulation of reproduction and reproductive behaviours. We mapped activity in the POA evoked by 17,20β-P exposure using the immediate-early gene c-fos. We found that a population of ventral POA neurones close to kisspeptin2 (kiss2) neurones that appear to have important roles in reproduction was activated by 17,20β-P exposure, suggesting that these activated neurones are important for the 17,20β-P response. Next, we investigated the distribution of androgen receptor (ar) in the POA and its relationship with 17,20β-P-responsive and kiss2 neurones. We found that ar is widely expressed in the ventral POA, whereas it is only expressed in approximately 10% of 17,20β-P-activated neurones. On the other hand, it is expressed in almost 90% of the kiss2 neurones. Taken together, it is possible that ar expressing neurones in the ventral POA, most of which were not labelled by c-fos in the present study, may at least partly account for androgen effects on responses to primer pheromones; the ar-positive kiss2 neurones in the ventral POA may be a candidate. These results offer a novel insight into the mechanisms underlying male-specific information processing of 17,20β-P in goldfish.

From bench to bedside: bipolar androgen therapy in a pilot clinical study.

Prostate cancer remains a leading cause of cancer death in Europe and the United States and is an emerging problem in Asia despite significant improvements in available treatments over the last few decades. Androgen deprivation therapy (ADT) has been the core treatment of advance-staged disease since the discovery of prostate cancer's androgen dependence in 1941 by Huggins et al.1 Options for initial medical treatment include gonadotropin-releasing hormone analogues such as leuprolide (LHRH agonist) and degarelix (LHRH antagonist) and androgen receptor (AR) binding agents such as bicalutamide. Although most patients will initially respond to either surgical or medical castration, there is almost always progression to castration-resistant prostate cancer (CRPC) necessitating treatment with more novel agents.2 However, even drugs such as abiraterone and enzalutamide, two next-generation agents used commonly in metastatic CRPC, have failed to demonstrate persistent efficacy in most patients.34

Abstract IA11: The role of bone microenvironment in the lethal progression of prostate cancer

Abstract Lethal prostate cancer (PCa) is clinically characterized by bone-homing and bone- forming metastases, and bone is frequently the initial organ involved in castrate-resistant progression. These clinical observations led investigators to develop bone-tropic radiopharmaceuticals to treat men with advanced PCa. The ability of the bone-homing alpha emitter (RAD-223) to prolong progression free and overall survival establishes the therapeutic relevance of targeting bone 1. The long accepted paradigm of PCa progression has been dominated by the model of evolution from androgen dependence to androgen independence under the selective pressure of castration. Thus, Src family kinases (SFKs) were investigated experimentally and clinically for the treatment of putative “androgen independent” PCa, as SFKs play a role in both tumor growth and bone turnover. The roles of SFKs in migration, invasion, survival of tumor cells, and osteoclast and osteoblast function in tumor microenvironment were supported by numerous clinical and laboratory observations 2, 3. Despite promising phase I /II reports of dasatinib, a multikinase and potent SFK inhibitor, in men with advanced PCa 4, 5, a phase III trial failed to yield the expected survival advantage 6. The result of this trial highlights the difficulties in translating data from cell line models to biologically heterogeneous clinical PCa and in transitioning from phase I/II to phase III clinical trials. The efficacy of dasatinib was not observed likely due to persistent AR signaling, which remains the primary driver of progression in bone by the transition from paracrine to intracrine androgen signaling 7. Based on these considerations, we are testing the efficacy of an androgen biosynthesis inhibitor in combination with dasatinib to better understand the relevance of mechanism(s). A co–clinical investigational strategy is being applied to better define biomarkers to predict clinical efficacy. These studies also highlight the importance of preclinical work that utilizes models that better reflect the heterogeneity of PCa. This problem is partly overcome by patient- derived xenografts (PDX) 8, 9 10. These models reflect many aspects of PCa in bone and can be used to identify therapy targets and better identify both mechanisms of drug efficacy and development of resistance. Studies in PDX have led to identification of FGF signaling as a therapy target 11. Additional studies in PDX implicated the tumor microenvironment in the striking bone responses observed with cabozantinib 13. These observations led us to propose a model of prostate cancer progression founded on the hypothesis that cancer progresses from being an endocrine-driven cancer (Dihydrotestosterone-dependent) to paracrine/intracrine driven (microenvironment-dependent), and then finally to a cell autochthonous phase 12. Continued studies should lead to decisions on therapy treatment that are based on an understanding of markers that predict disease progression. This new paradigm will also result in more effective therapy combinations.