1320 ZOLEDRONIC ACID (ZOL) ENCAPSULATED INTO LIPOSOMES AND NANOPARTICLES: ENHANCED ANTITUMOR ACTIVITY IN 3D- PROSTATE CARCINOMA SPHEROIDS

Vol. 189, No. 4S, Supplement, Monday, May 6, 2013

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Prostate Cancer: Basic Research (VI) Moderated Poster Session 49 Monday, May 6, 2013

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1318 HYPOXIC MICROENVIRONMENT PROMOTES PI3K/AKT/MTOR SIGNALING PATHWAYS IN HUMAN CASTRATION RESISTANT PROSTATE CANCER Yota Yasumizu*, Takeo Kosaka, Yasumasa Miyazaki, Eiji Kikuchi, Akira Miyajima, Mototsugu Oya, Tokyo, Japan INTRODUCTION AND OBJECTIVES: Recently we demonstrated that the activated PI3K-Akt-mTOR signaling pathway induced by androgen deprivation therapy or docetaxel explains at least in part the aggressiveness in CRPC. However microenviromental regulation of PI3K/Akt signaling pathways or the efficacy of PI3K/Akt inhibitors in hypoxic condition has not been fully characterized yet. METHODS: Human CRPC cell lines: C4-2 and C4-2AT6 were used in this study. The C4-2AT6 cell line was established from C4-2 cells grown in androgen ablated serum for six months. We examined the change of phosphorylated Akt (pAkt) and phosphorylated S6 expression under hypoxic condition (1-5%O2) using hypoxic chamber. We explore the efficacy of PI3K/Akt/mTOR inhibitors: NVP-BEZ235 and everolimus in hypoxic condition. Epithelial mesenchymal transition (EMT) related markers or invasive activity under normoxic and hypoxic conditions was quantified. RESULTS: We investigated the expression of phosphorylated pAkt and pS6 in C4-2AT6 cultured under normoxia or hypoxia. Although under normoxia C4-2AT6 expressed elevated pAkt and pS6 accompanied by elevated HIF-1a expression, 1-5% hypoxic condition further induced pAkt and pS6 expression in C4-2AT6. These results indicates HIF-1 or Akt, which is induced in aggressive PCa, are induced moreover under hypoxic condition. Next we examined the inhibitory effect of PI3K/Akt/mTOR inhibitor. The expression of pAkt and pS6 under hypoxia of 1% O2 was significantly inhibited by at the dose of 500nM NVP-BEZ235 for 24 hr culture period. The expression of pAkt and pS6 were inhibited by NVP-BEZ235 at the dose dependent manner. These results suggested that NVP-BEZ235 inhibited PI3K/Akt/ mTOR signaling pathway under hypoxia as well as normoxia despite the further up-regulated pAkt under hypoxia. The cytotoxic effect of NVP-BEZ235 under hypoxia was examined by direct cell count. The number of cells under hypoxia of 1% O2 was 4.08 ⫾ 0.75 ⫻ 105/ ml and the number of cells treated with 500nM NVP-BEZ235 under hypoxia of 1% O2 was 2.72 ⫾ 0.82 ⫻ 105/ ml. The difference of the cell number was significant (p⬍0.05). The morphological change of cancer cells from mesenchymal to epithelial was induced under hypoxia at 24 hr, suggesting the induction of EMT, although E-cadherin expression was not significantly changed for 24 h culture period. CONCLUSIONS: Hypoxic microenvironment promotes PI3K/ Akt/mTOR signaling pathways and morphological change in human CRPC. Source of Funding: None

1319 A NEAR-INFRARED IMAGING AGENT IDENTIFIES PROSTATE SPECIFIC MEMBRANE ANTIGEN POSITIVE CELLS DURING LAPAROSCOPIC SURGERY Brian Neuman*, John Eifler, Mark Castanares, Wasim Chowdhury, Ying Chen, Martin Pomper, Ronald Rodriguez, Baltimore, MD INTRODUCTION AND OBJECTIVES: Depending on the stage there is approximately a 4-54% positive surgical margin (⫹SM) following prostatectomy. Patients with ⫹SM face the associated expense and impaired functional outcomes of salvage radiotherapy. To circumvent this, a low-molecular weight urea-based near infrared (NIR) fluorophore

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(YC-27) was developed to target the prostate specific membrane antigen (PSMA) for use in intraoperative imaging. PSMA is a transmembrane protein expressed in normal prostate and upregulated in cancer. PSMA expression correlates with Gleason score, advanced stage and PSA. We optimized dosing and timing in a small animal model for laparoscopic resection of PSMA positive tumors. Secondly, we demonstrate the feasibility of applying such a system in a porcine model. METHODS: PSMA positive and negative tumors were established contralaterally on the flank of athymic male nude mice. YC-27 was administered intravenously at varying doses (9.5, 19.1, 39.7 [ug/ Kg]) and sequentially imaged using a custom built laparoscopic system to obtain probe kinetics (1, 2, 3, 4, 6, 8, 10, 24 hrs post IV injection). Arbitrary pixel count was used to evaluate dosing and timing for signal to noise ratio. NIR guided laparoscopic resection was performed on mice with established PSMA positive and negative tumors 6 hrs post injection of 39.7 ug/Kg YC-27. In a porcine model, prestained human xenografts generated in a murine host were implanted behind the peritoneum of the abdominal cavity, and exploratory NIR laparoscopy was performed. In a porcine model, the kidney was observed with our laparoscopic NIR system after intravenous YC-27 administration. RESULTS: In a murine model, the highest signal to noise ratio was determined to occur 6 hrs after injecting 39.7 ug/Kg YC-27. Using these parameters, NIR guided laparoscopic surgery facilitated full resection of the xenograft. In a porcine model, exploratory laparoscopy with our NIR system allowed detection of a prestained tumor implanted behind the peritoneum. In another experiment, strong fluorescent signal was observed from the porcine kidney immediately after IV injection of YC-27 which may be attributed to specific binding to PSMA, renal clearance, or a combination of both. CONCLUSIONS: YC-27 allows intraoperative localization of PSMA in real time using a prototype NIR guided laparoscopic surgical system. Pilot studies in large animals demonstrated excellent potential for clinical translation. Source of Funding: Patrick C. Walsh Prostate Cancer Research Fund.

1320 ZOLEDRONIC ACID (ZOL) ENCAPSULATED INTO LIPOSOMES AND NANOPARTICLES: ENHANCED ANTITUMOR ACTIVITY IN 3D- PROSTATE CARCINOMA SPHEROIDS Johannes Schmidt, Homburg, Germany; Giuseppe De Rosa, Naples, Italy; Joern Kamradt, Kerstin Junker, Michael Stoeckle, Homburg, Germany; Michele Caraglia, Naples, Italy; Gerhard Unteregger*, Homburg, Germany INTRODUCTION AND OBJECTIVES: Zoledronic acid (ZOL) is currently limited for the treatment of bone metastases in hormoneresistant prostate cancer. The use of nanomedicine is one promising strategy to improve the availability of drugs within the tumor tissue. To investigate the benefit of such approach, we used nanovectors encapsulating ZOL on 3D-spheroids from prostate carcinoma cells. METHODS: Stealth liposomes (PGNP_ZOL) and self-assembly nanoparticles (NP_ZOL) were prepared as already described. The prostate carcinoma cell lines PC-3, DU-145 and LNCaP and tumorassociated fibroblast (TAF) were cultivated as monolayers and as Spheroids in 96-well MTP and incubated with the drugs for 120 h. Changes in growth behavior and toxicity (apoptosis/necrosis) in 2D/3Dcultures was quantified by modifications of standard assays. RESULTS: Standard assays revealed a significant enhancement of antitumor activity of NP_ZOL or PGNP_ZOL as compared to the free drug in DU-145 and LNCaP, but these effects were less pronounced in PC-3 cells. The efficacy of NP_ZOL and PGNP_ZOL was caused only by inhibition of cell proliferation (BrDU) since apoptosis and necrosis were not detected. The average IC50 values in the tumor cells ranged from 10-20␮M/L whereas fibroblasts were more sensitive (IC50 2-5␮m/L ZOL).

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CONCLUSIONS: Our results confirm the hypothesis that the use of nanomedicine enables the extension in therapeutic applications of drugs such as bisphosphonates. Homo-/heterologeous 3D-spheroids offer advanced models mimicking the microenvironment within a tumor tissue thus bridging the gap between cell culture and live tissue. Moreover, the rapid generation of single-tumor spheroids allows for a high-throughput cell function and toxicity analysis. Source of Funding: University.

1321 THE PROSTATE CANCER TUMOUR SUPPRESSOR MICRO RNA 1, REGULATES PLEXIN B1 EXPRESSION Adebiyi Damola*, Magali Williamson, London, United Kingdom INTRODUCTION AND OBJECTIVES: It is known that microRNAs can be tumour suppressors and recent studies have shown that microRNAs have this function in prostate cancer. The main mechanism of action of MicroRNAs is by binding to the 3⬘ untranslated region (UTR) of messenger RNAs and inhibiting their translation. Plexin B1 is a transmembrane receptor for semaphorin 4D that act as chemotactic cues for cell migration. There is overexpression and mutation of Plexin B1 in prostate tumours that suggests Plexin B1 has a role in prostate cancer and is a potential target for therapy perhaps by microRNAs mimics. Our study aimed to find out whether Plexin B1 is regulated by microRNAs and identify the microRNAs that could carry out this function. METHODS: In order to investigate the effects of microRNAs on plexin B1, a plexin B1 3⬘UTR luciferase reporter construct, termed Luc3⬘UTR was cloned and transfected into prostate cancer cell lines known to express plexin B1.The effect of the presence of the 3⬘UTR on Luciferase expression was determined by comparing the Luciferase activity of cells transfected with the Luc-3⬘UTR to control luciferase construct. To identify microRNA that potentially regulate Plexin B1 real time quantitatitve PCR (QPCR) was performed on transcriptome arrays containing cDNA from Hela cells transfected with cancer microRNA mimics or controls using SYBR green dye. Relative levels of expression of Plexin B1 in Hela cells were quantified with the mimics relative to untransfected controls and normalised using the housekeeping gene GAPDH. RESULTS: There was a difference in the effect of Luc- 3⬘UTR compared to the Luc-control across the cell lines with reduced luciferase activity in cells transfected with Luc- 3⬘UTR compared to control. This suggests a regulatory role for microRNAs by binding to 3⬘UTR ends of Plexin B1 mRNAs. The QPCR transcriptome array revealed microRNA 1 as a potential negative regulator of plexin B1 expression as shown in the graph below. CONCLUSIONS: The results suggests microRNAs do control the expression of plexin B1 and that in particular microRNA 1 may be involved. Studies have shown that microRNA 1 acts a tumour suppressor in prostate cancer and that it is down regulated in prostate cancer. MicroRNA 1 may be a therapeutic option in prostate cancer subtypes linked to plexin B1.

Source of Funding: None

Vol. 189, No. 4S, Supplement, Monday, May 6, 2013

1322 LIN28 PROMOTES GROWTH OF PROSTATE CANCER CELLS AND ACTIVATES THE ANDROGEN RECEPTOR Ramakumar Tummala*, Nagalakshmi Nadiminty, Yezi Zhu, Wei Lou, Christopher P. Evans, Allen C. Gao, Sacramento, CA INTRODUCTION AND OBJECTIVES: Lin28 is essential for stem cell viability and pluripotency. Recently Lin28 expression has been linked to several malignancies and its mechanisms of action proposed to be both let-7-dependent and –independent.It is a master regulator of let-7 miRNA biogenesis as well as a regulator of translation of a multitude of eukaryotic proteins. This study tested the hypothesis that Lin28 is overexpressed in prostate cancer (CaP) and that it activates the androgen receptor (AR) and promotes growth of CaP cells. METHODS: Human clinical CaP samples were analyzed for the expression of Lin28 by qRT-PCR, Western blotting and immunohistochemistry.Lin28 was transfected into a panel of CaP cell lines and growth was monitored. LNCaP cells stably expressing Lin28 were generated and growth was examined. Clonogenic ability of CaP cells expressing Lin28 was determined by colony formation and soft agar assays. Increase in expression of the AR and subsequent increase in transcription of AR-target genes were analyzed by qRT-PCR, luciferase assays and ELISA. LNCaP cells stably expressing Lin28 were injected into nude mice and tumorigenesis was monitored. RESULTS: Lin28 was found to be overexpressed in clinical CaP compared to the benign tissues. Overexpression of Lin28 enhanced while downregulation of Lin28 reduced growth of CaP cells. Lin28 enhanced the ability of CaP cells to form colonies in anchoragedependent and –independent manners. LNCaP cells stably expressing Lin28 exhibited significantly higher tumorigenic ability in vivo.Furthermore, Lin28 induces expression of the AR and its target genes such as PSA and NKX3.1. CONCLUSIONS: Lin28 promotes growth of CaP cells in vitro and in vivo. Lin28 induces expression and activation of the AR. Our findings suggest an important role of Lin28 in CaP development and activation of the AR axis. Source of Funding: This work was supported in part by National Institutes of Health Grants CA140468, CA118887, DOD PCRP PC080538 (A.C.Gao) and DOD PCRP PC100502 (N. Nadiminty).

1323 RNA-BINDING MOTIF PROTEIN 3 ATTENUATES THE STEM CELL-LIKE PROPERTIES OF PROSTATE CANCER CELLS BY INTERFERING WITH CD44 VARIANT SPLICING Yu Zeng*, Dong Gao, Dana Wodzenski, Takumi Shiraishi, Naoki Terada, Jun Luo, Robert Getzenberg, Prakash Kulkarni, Baltimore, MD INTRODUCTION AND OBJECTIVES: Stress response pathways appear to play an important role in cancer evolution. RNA-binding motif protein 3 (RBM3) is a stress-response protein that is downregulated under certain micro-environmental stress effects. Further, RBM3 expression in primary tumors is inversely correlated with poorer prognosis suggesting that RBM3 may suppress tumor progression. The goal of this study was to investigate the effects of RBM3 on stem cell-like features of prostate cancer cells. METHODS: RBM3 mRNA expression was detected by RTPCR. The normal prostate cell line, PrEC, was sorted using CD133 by flow cytometry. PC3 cells were transfected with pCMV6-RBM3 vector. Soft agar clonogenic assay and prostasphere assay were performed to evaluate stem cell-like features. The expressions of stem cell-related genes were determined using PCR arrays. PC3-RBM3 and PC3-GFP clones were subcutaneously inoculated in nude mice and tumor growth were compared. RESULTS: RBM3 expression was low in human fetal tissues and in the CD133⫹ PrEC cells. In human prostate cancer, the expres-