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adenocarcinomas until 12 months. CONCLUSIONS: Inactivation of GSTP1 in mice accelerates MYC-

induced prostate carcinogenesis. This study is the first to provide direct evidence that GSTP1 can function as a tumor suppressor in the prostate.

Source of Funding: None

515TARGETING INSULIN-LIKE GROWTH FACTOR-1 RECEPTOR WITH ANTISENSE OLIGONUCLEOTIDES IN PROSTATE CANCER

Junya Furukawa*, Vancouver, BCCanada; Christopher J Wraight, Toorak, Australia; Brett P Monia, Carlsbad, CA; Michael E Cox, Martin E Gleave, Vancouver, BC, Canada

INTRODUCTION AND OBJECTIVE: Initiating as an androgen-dependent adenocarcinoma, prostate cancer (PCa) gradually progresses to a castrate-resistant disease (CRPC) following androgen deprivation therapy (ADT) with a propensity to metastasize. Altered expression of insulin-like growth factor (IGF) axis components have been consistently found in PCa. The action of IGFs is mediated through IGF-I receptor (IGF-IR) and modulated by IGF binding proteins (IGFBPs). We hypothesize that increased expression and/or responsiveness of IGF-IR may promote CRPC progression in patients undergoing ADT. In this study, we assessed ATL1101, a 2’-MOE-modified antisense oligonucleotide (ASO) targeting human IGF-IR, with regard to potency and anti-cancer activity of IGF-1R ASO treatment on androgen-responsive (LNCaP) and independent (PC3) PCa cells in vitro and in vivo.

METHODS: IGF-IR mRNA and protein expression in ATL1101- and control oligonucleotides (ODN)-treated LNCaP and PC3 cells were observed by QT-PCR and western blotting. The effect of IGF-1R ASO on LNCaP and PC3 cell growth and apoptosis in vitro was examined by MTT assay and flow cytometry. Effects of the IGF-1R ASO on various putative targets were evaluated using western blotting. For in vivo xenograft studies, LNCaP or PC-3 cells were inoculated s.c. in the flank region of athymic nude mice. Mice were castrated and randomly treated with ATL1101 or control ODN injected i.p. Tumor volume and serum prostate-specific antigen (PSA) were evaluated. Pharmacodynamic activity in vivo was assessed using immunologic analysis of harvested tumor tissues.

RESULTS: We observed dose- and sequence-specific suppression of IGF-IR mRNA and protein expression in ATL1101-treated LNCaP and PC3 CaP cells in vitro. Suppressed IGF-IR expression correlated with decreased proliferation and increased apoptosis of PC3 cells under standard culture conditions and increased apoptosis of LNCaP cells under androgen-deprived culture conditions. Compared to control ODN, ATL1101 significantly suppressed PC3 tumor growth as a monotherapy in murine xenografts. Similarly ATL1101 significantly delayed CR progression of LNCaP xenografts as measured by tumor growth and serum PSA levels. We confirmed that suppression of IGF-IR expression correlated with decreased tumor growth in vivo.

CONCLUSIONS: This study reports the first preclinical proof-of-principle data that this novel IGF-IR ASO selectively suppresses IGF-1R expression, suppresses growth of CRPC tumors and delays CRPC progression in vitro and in vivo.

Source of Funding: Antisense Therapeutics, Ltd. and grants from National Cancer Institute of Canada.

516IMPROVED PREDICTION OF BIOCHEMICAL RECURRENCE AFTER RADICAL PROSTATECTOMY BY DNA MICROARRAYS

Juan Morote*, Barcelona, Spain; Jokin del Amo, Bilbao, Spain; A Borque, Zaragoza, Spain; Jacques Planas, Carles Raventos, Barcelona, Spain; C Allepuz, Zaragoza, Spain; J Larrinaga, Vitoria, Spain; R Llarena, Bilbao, Spain; JM Campa, Vitoria, Spain; MJ Viso, Zaragoza, Spain; D Tejedor, L Simon, A Martinez, Bilbao, Spain; LA Rioja, Zaragoza, Spain

INTRODUCTION AND OBJECTIVE: Single Nucleotide Polymorphisms (SNPs) capacity for predicting cancer initiation and

progression can be increased combining multiple SNPs. Proscan is a DNA microarray able to detect 83 SNPs associated to the risk and progression of prostate cancer (PCa). The aim of our study was to explore whether the SNP profile provided by Proscan can improve the prediction of biochemical progression (BP) after radical prostatectomy (RP).

METHODS: DNA was collected from peripheral blood of 311 patients undergoing RP followed for at least 5 years or until BP. Patients’ SNP profile was obtained by amplifying the genes of interest in 6 multiplex-PCRs, followed by fluorescent labelling and hybridisation to Proscan microarray. Baseline clinicopathologic variables (CVs) were recorded: onset age, PSA, clinical stage, specimen Gleason score, pathological stage, tumor volume and margin status. In order to predict BP, two stepwise logistic regression models were developed; the first used CVs only and the later included both CVs+SNPs. Accuracy of the models was assessed by calculating the AUC-ROC with 95% CI, R2 Nagelkerke, sensitivity, specificity, and positive likelihood ratio.

RESULTS: Overall rate of BP was 27.7%. Models were developed on the training cohort (median age 64.2 years, median PSA 11.6 ng/ml). The model with the best fit combined 7 co-variables: 4 SNPs located at TP53, TMPRSS2, LPL and CYP1A1 genes and 3 CVs: PSA, Gleason score, margin status. Predictive power of the model based on SNPs+CVs (R2=0,382; AUC=83%; Se=42%; Sp=95%; LR+=8,5) was significantly better (p=0.01) than the one based on CVs (R2=0,283; AUC=78%; Se=23%; Sp=95%; LR+=4.9). Positive and negative predictive values were also higher when the model used both CVs+SNPs. Model performance was evaluated on a validation cohort (median age 62.6 years, median PSA 13.2 ng/ml). The performance of the model was closely similar to that on the training cohort (AUC=77%; Se=46%; Sp=93%; LR+=7). No significant differences were found between AUC derived from training and validation cohorts (p=0.921). A nomogram based on CVs and SNPs was constructed.

CONCLUSIONS: Prediction of biochemical recurrence after radical prostatectomy based on clinicopathological variables can be significantly improved by including SNP profiles. Addition of SNP profiles to currently available clinical nomograms can help to accurately identify patients at high risk of biochemical progression. Further validation and its resulting nomogram in larger series would strengthen these conclusions.

Source of Funding: None

517NF- B ACTIVATION FACILITATES PROSTATE CANCER PROGRESSION

Vladimir Yutkin*, Shay Tayeb, Eli Pikarsky, Jerusalem, Israel

INTRODUCTION AND OBJECTIVE: Nuclear factor kappa B (NF- B) is a collective designation for a family of highly regulated dimeric transcription factors. Recent works suggest that NF- B is involved in several stages of carcinogenesis in different tissues, including prostate cancer (PCa) with aberrant sustained activation in various stages of tumorigenesis. In this project we explore the role of NF- B in PCa development using in-vivo model in genetically modified mice.

METHODS: We employ the ProbasinCre/PTENlox/lox model of murine prostate cancer (control group). To study the effect of NF-B activation we bred these mice with R26-lox-stop-lox-IKK mice. To

a second study group we added a tetracycline regulated I B super-repressor, thus blocking NF- B activity. We sacrificed these mice on different ages to study the prostate phenotype both in heterozygous (PTENlox/+) and in homozygous (PTENlox/lox) genotype. We compared the resultant phenotype of both over-active and under-active NF- B to the classic ProbasinCre/PTENlox/lox phenotype. We also performed the immunohistochemical and biochemical studies of the prostate tissues to explore the function of NF- B family, apoptosis and proliferation activity.

RESULTS: In the control group we observed development of high grade prostate intraepithelial neoplasia (HG PIN) at 3-4 months and focal invasive carcinoma at 6-8 months. We did not see metastatic disease up to year of observation. In addition male mice were infertile after age of 5-6 months. Addition of constitutive NF-kB activation leads to striking enhancement of the cancerous phenotype - both macroscopic

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and microscopic. At 3 months the entire prostate was infiltrated by carcinoma and at 6 months there were large pelvic masses of invasive PCa. Immunohistochemisry with p65 showed intensive positive nuclear staining in areas of cancer. However, blocking NF- B did not change the phenotype relative to the controls, with only one exception: normal fertility.

CONCLUSIONS: In these two in-vivo models we demonstrate that up-regulating NF- B signaling greatly augments cancer phenotype, probably by virtue of enhancing cancer progression, but not initiation. These findings suggest that inflammatory cytokines that upregulate NF-B could promote prostate cancer growth. Moreover, NF- B inhibition

could be an effective therapeutic modality in patients with PCa.

Source of Funding: Research grant from “Prostate Cancer Research Foundation”, PCRF

518INHIBITION OF THE AQUAPORIN WATER CHANNELS INCREASES THE SENSITIVITY OF PROSTATE CANCER CELLS TO FREEZING INJURY.

Mohamed Ismail, Jr*, Richard Morgan, Sr, John Davies, Sr, Hardev Pandha, Sr, Guildford, United Kingdom

INTRODUCTION AND OBJECTIVE: Prostate cryotherapy has emerged as an alternative minimally invasive treatment option for prostate cancer. Complete ablation of cancer tissue some times fails and results in disease recurrence. The aquaporin (AQP) family of water channels are intrinsic membrane proteins that facilitate selective water and small solutes movement across the plasma membrane. In this study we have assessed the potential role of AQPs inhibition in sensitising tumour cells to cryotherapy to enhance the antitumour efficacy.

METHODS: A novel model of freezing prostate cancer cells was established using Endocare cryo-system. Prostate cancer cells were cooled to 0, -5 and -10°C for 10 minutes and thawed in 55°C water bath. The expression of AQP1, AQP3 and AQP9 in response to freezing was examined using quantitative polymerase chain reaction (q-PCR) and western blot analysis. Prostate cancer cells were exposed to freezing injury in the presence or absence of the aquaporin inhibitor mercuric chloride (HgCl2) and cell survival was assessed after freezing using colorimetric assay. Small interfering RNA duplex (siRNA) was used to specifically suppress AQP3 and cell survival was assessed following the treatment.

RESULTS: Prostate cancer cells express AQP1, AQP3 and AQP9. There was a significant increase in AQP3 mRNA and protein expression levels in the immediate post freeze period followed by reduction to the untreated level by 24 hours post freezing. Inhibition of AQPs by HgCl2 increased the sensitivity of DU145 cells to freeze injury and there was a complete loss of cell viability at -10°C (P<0.01). qPCR and Western blot analysis showed that AQP3 silencing caused a significant reduction in AQP3 mRNA and protein levels. AQP3 knocked down cells were more sensitive to freeze injury compared to control cells (P<0.001).

CONCLUSIONS: We demonstrated that cryotherapy alone may fail to kill prostate cancer cells completely and sensitising agent may be needed to improve the long term clinical outcome following cryotherapy. We demonstrated that AQP3 is involved directly in cryoinjury. Inhibition of AQP3 increases the sensitivity of prostate cancer cells to freeze injury. Further research in the clinical application of AQP inhibitors is needed.

Source of Funding: The Prostate Project Foundation

519IMMUNOSTIMULATORY CPG-DNA AND PSA-PEPTIDE VACCINATION ELICITS PROFOUND CYTOTOXIC T CELL RESPONSES

Tobias Maurer*, Antonio Juan Aguilar-Pimentel, Christos Pournaras, Mark Thalgott, Antje Heit, Juergen E Gschwend, Hubert Kuebler, Roman Nawroth, Munich, Germany

INTRODUCTION AND OBJECTIVE: Currently, immunotherapy as treatment option is under investigation in advanced prostate cancer.

Here we examine the potency of CpG-DNA oligonucleotides (ODN) to boost cytokine responses and costimulatory molecule regulation on murine bone marrow derived dendritic cells (mBMDC) as well as the potency of a prostate specific antigen (PSA)-peptide based vaccine in combination with CpG-DNA to elicit PSA-peptide specific cytotoxic T cell (CTL) responses in C57/BL-6 mice.

METHODS: mBMDC were cultivated and stimulated with stimulatory CpG-DNA-ODN (1668: 5´-TCCATGACGTTCCTGATGCT-3´) and non-stimulatory control-ODN (1720: 5´-TCCATGAGCTTCCTGATGCT-3´), subsequently analysed for expression of the costimulatory molecules CD40 and CD86 by FACS. Induction of the proinflammatory Th1-cytokines IL-6 and IL-12 was determined after in vitro stimulation of mBMDC with 1668- and 1720-ODN by ELISA. For induction of PSA-peptide specific CTL, C57/BL-6 mice were immunized with PSA-peptide 65-73 (HCIRNKSVI) alone or in combination with 1668 or 1720-ODN i.v. on day 1. Cytotoxic potential was demonstrated by in vivo lysis of highly fluorescence-labeled PSA-peptide-loaded splenocytes in comparison to unloaded low flourescence-labeled splenocytes.

RESULTS: CpG-ODN significantly increases production of IL-6 and IL-12 in mBMDC compared to control-ODN that could not elicit relevant cytokine levels (p<0.001). Regulation of costimulatory molecule expression on mBMDC is CpG-dependent and increases with higher concentrations of immunostimulatory CpG-DNA. The same concentrations of control-ODN showed no significant activation. Induction of PSA-peptide specific CTL responses in mice immunized with PSA-peptide and CpG-DNA were significantly higher (28.1%, 47.0% after 40h, 88h) than of PSA-peptide and control-ODN immunized mice (9.1%, 16.8%) or PSA-peptide only vaccination (3.2%, 9.8%).

CONCLUSIONS: Here we show that CpG-DNA can act as potent adjuvant for vaccination therapies. In combination with the immunodominant PSA-peptide 65-73 (HCIRNKSVI) it can elicit profound PSA-peptide specific cytotoxic T cell responses and could therefore be of value in peptide or protein based immunization therapies against prostate cancer.

Source of Funding: KKF TU München; Siegfried-Gruber-Stiftung, München

520GLOBAL HISTONE MODIFICATIONS AS A PROGNOSTIC MARKER FOR BIOCHEMICAL RECURRENCE IN PATIENTS UNDERGOING RADICAL PROSTATECTOMY

Patrick J Bastian*, Munich, Germany; Johannes von der Gathen, Philip Kahl, Ines Gütgemann, Stefan C Müller, Alexander von Ruecker, Jörg Ellinger, Bonn, Germany

INTRODUCTION AND OBJECTIVE: Epigenetic alterations such as DNA methylation and histone modifications play important roles in carcinogenesis. It has been recently suggested that gobal histone modification patterns are independent predictors of cancer recurrence. In this study, we used immunohistochemistry to evaluate the patterns of histone H3 lysine 4 (H3K4) and histone H3 lysine 9 (H3K9) methylation in prostate cancer (PCA).

METHODS: We examined the global level of H3K4 mono- (H3K4me1), di- (H3K4me2) and tri (H3K4me3) methylation and H3K9 mono- (H3K9me1), di- (H3K9me2) and tri (H3K9me3) methylation using a tissue micro array [including patients with clinically localized prostate cancer (PCA; n=113), benigne prostate hyperplasia (n=17) and normal prostate tissue (n=6)]. Sections were scored according the staining intensity and the proportion of cells showing nuclear staining.

RESULTS: Staining of H3K9me2 (p<0.001) and H3K9me3 (p=0.030) was less intense in PCA compared to non-malignant prostate tissue, whereas staining of H3K9me1 (p=0.062), H3K4me1 (p=0.122), H3K4me2 (p=0.922) and H3K4me3 (0.375) were similar in PCA and non-malignant prostate tissue. Correlations between global histone levels and preoperative PSA (H3K4me2, H3K9me2, H3K9me3; p=0.034-0.013), pT-stage (H3K9me2, p=0.049), pN-stage (H3K4me1, H3K4me2, H3K4me3, H3K9me2; p=0.016-0.001) and Gleason Score (H3K4me2, H3K4me3, H3K9me1, H3K9me2; p=0.034-0.001). A high level of