1281 ANTI-TUMOR ACTIVITY OF SULFATED HYALURONIC ACID, A HYAL1 HYALURONIDASE INHIBITOR, IN PROSTATE CANCER CELLS

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cells induced by EGF may lead to new therapeutic considerations for advanced prostate cancer.

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1280 WNT SIGNALING PROTEIN (WISP2/CCN5) STIMULATES ANGIOGENESIS AND INVASION IN PROSTATE CANCER Yasunobu Hashimoto*, Rajendra Singh, Daniel Mun˜oz, Bal Lokeshwar, Miami, FL

Source of Funding: None

1279 CLU INHIBITION USING OGX-011 IS A NEW ADJUVANT THERAPEUTIC STRATEGY FOR HSP90 INHIBITION IN PROSTATE CANCER Francois Lamoureux*, Vancouver, Canada; Min-Jean Yin, San Diego, CA; Amina Zoubeidi, Martin Gleave, Vancouver, Canada INTRODUCTION AND OBJECTIVES: Prostate cancer (PCa) responds initially to anti-androgen therapies, however, progression to castration resistant disease (CRPC) frequently occurs. Several small molecule inhibitors of HSP90 show promise in CRPC and other cancers. However, most HSP90 inhibitors (17-AAG or PF-04928473 and its prodrug PF-04929113) trigger the elevation of HSPs (HSP90, 70, 27 and clusterin), which lead to tumor cell survival and treatment resistance. We hypothesized that targeting clusterin (CLU) using siRNA or the antisense drug, OGX-011, may enhance HSP90 inhibitors-induced cell death in PCa. METHODS: Inducible and constitutive CLU and other HSP mRNA and protein levels were measured by real-time RT-PCR and immunoblot assays. The combination of OGX-011 with PF-04928473 or 17-AAG was evaluated in vitro on LNCaP and PC3 cells growth and apoptosis. The HSP90 inhibitor PF-04929113 was evaluated in combination with OGX-011 in vivo in athymic mice bearing castration resistant LNCaP xenografts, while the combination of OGX-011 with 17-AAG was evaluated in PC3 xenografts. RESULTS: In prostate tumor cell lines, PF-04928473 and 17AAG induced expression of HSPs in a dose and time dependent manner, and especially CLU at RNA and protein level, by increasing HSF-1 nuclear translocation and transcription activity. In vitro, OGX011 synergistically enhanced the activity of HSP90 inhibitors on cell growth and apoptosis with increased sub-G1 fraction and PARP cleavage. These biologic events were accompanied by decreased expression of HSPs, Akt, AR and PSA, and HSF-1 transcriptional activity. In vivo, OGX-011, administered 3 times a week (IP, 15mg/kg), potentiated the effect of orally administered HSP90 inhibitors (PF-04929113: 25mg/kg; 17-AAG: 50mg/kg), significantly inhibiting tumor growth by 80% and prolonging survival in PC3 and castrate resistant LNCaP xenograft model compared to the HSP90 inhibitors alone. CONCLUSIONS: These results indicate that HSP90 inhibitormediated induction of CLU expression can be attenuated by OGX-011, with synergistic effects on delaying progression of CRPC. Source of Funding: This work was supported by the Canadian Institutes of Health Research fellowship and Association pour la Recherche sur le Cancer, France.

INTRODUCTION AND OBJECTIVES: Angiogenesis and invasion are essential attributes of metastatic cancer cells. The WntInduced Signaling Protein-2 (Wisp-2 /CCN5) is a secreted protein implicated in modification of extracellular matrix, invasion, and angiogenesis. The regulation of Wisp-2 and its function in CaP is poorly explored although it is highly expressed in advanced CaP cells. We discovered recently that a pro-inflammatory chemokine, Interleukin-8 (IL-8) strongly modulates Wisp2 expression in CaP cells. Since chronic inflammation is thought to be significant in CaP metastasis and IL-8 is a major effecter of inflammatory pathway, we investigated the physiological consequence of Wisp-2 modulation in CaP cells. We hypothesised that Wisp-2 is a down-stream effector of IL-8 induced angiogenesis and invasion, and is essential for metastatic progression of prostate cancer. METHODS: Quantitative PCR (qPCR) was used to determine the expression of Wisp-2 mRNA and other mRNA in prostate epithelial and cancer cells (e.g. RWPE-1, LNCaP, LAPC-4, DU145, and PC3). Wisp-2 mediated-VEGF secretion was determined by ELISA and endothelial tube formation assay using human vascular endothelial cells (HUVEC cells). Invasive activity and their attributes were determined using gelatin-zymography for matrix metalloproteinases (MMPs), Matrigel invasion assay and chemotaxis assay. RESULTS: Wisp2 RNA was strongly expressed in two highly metastatic CaP cell lines, DU145 and PC-3 and moderately in less invasive LAPC4 and LNCaP cells. Constitutive autocrine expression or external addition of IL-8 increased Wisp-2 mRNA by ⱖ15.2 times in LNCaP, and 7.4 times in LAPC4 cells. Depleting Wisp-2 by RNA interference caused 82% reduction of Wisp2 in DU145, and 75% in PC-3. Depletion of Wisp2 did not affect cell proliferation in DU145 and PC3 but it reduced expression of VEGF mRNA by 64% in DU145 and 43% in PC3. Secreted VEGF protein in cultures of DU145 and PC-3 cells showed a decrease of 62% in DU145 and 30% in PC3. Condition medium of Wisp-2 depleted DU145 cell cultures showed 20% decrease in proliferation and vessel formation activity in HUVEC cells. Further, Wisp-2 depletion in DU145 cells resulted in significant decrease in chemo-invasion activity (28%) and secretion of MMP-9 (25%). CONCLUSIONS: These results show that Wisp2 is down stream effector of IL-8 and is an important modulator, affecting extracellular matrix to stimulate angiogenesis and invasiveness in CaP cells. Suppression of Wisp-2 in CaP may reduce their metastatic potential. Source of Funding: VA MERIT Review VA5312.01 (BLL)

1281 ANTI-TUMOR ACTIVITY OF SULFATED HYALURONIC ACID, A HYAL1 HYALURONIDASE INHIBITOR, IN PROSTATE CANCER CELLS Ezekiel E. Young*, Travis Yates, Anaid Benitez, Luis E. Lopez, Vinata B. Lokeshwar, Miami, FL INTRODUCTION AND OBJECTIVES: Tumor cell-derived hyaluronidase HYAL1, which degrades hyaluronic acid (HA), promotes tumor growth and metastasis. In prostate cancer (CaP), HYAL1 expression in biopsy and prostatectomy specimens is an independent prognosticator for predicting disease progression. Sulfated hyaluronic acid (sHA) inhibits HYAL1 activity through a mixed inhibition mechanism. Antitumor activity of sHA has not been evaluated. In this study we evaluated effects of sHA on CaP cells. METHODS: Effect of sHA (0 – 40 ␮g/ml) on cell proliferation and apoptosis was examined in CaP cells (PC3-ML, LNCaP, LNAI, C4-2B, LAPC-4, DU145, RWPE1) by cell counting and Cell Death ELISA kit. Matrigel invasion and Boyden chamber assays were used to test the effect of sHA on invasive activity. Effect of sHA on signaling, apoptosis

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cascade, androgen receptor activity, VEGF, NF-kB activity, and HA receptor levels, was evaluated by immunoblotting, Q-PCR and reporter assays. Athymic mice bearing LNAI xenografts were treated with sHA (25 and 50 mg/kg) by intraperitoneal injection. RESULTS: sHA inhibited proliferation, motility and invasion in CaP cells. At IC50 for HAase activity inhibition (⬃ 10-mg/ml), sHA induced ⬎ 3-fold apoptosis and inhibited invasive activity of CaP cells, which could be partially reversed by HA-fragments. sHA induced caspase-3, -8, -9 activation, up-regulation of Fas, Fas-L, FADD, and DR4 and down regulated bcl-2, phospho-bad, bcl-XL, phospho-Akt, phospho-IkB, and phospho-AR. At IC50, sHA caused ⬎ 90% inhibition of NF-kB reporter activity, PSA-promoter activity and transcriptionally down regulated HA-receptors (CD44 and RHAMM) and VEGF. Effect of sHA could be reversed by overexpression of m-Akt. Down regulation of CD44 and RHAMM, together with sHA, synergistically inhibited cell proliferation and Akt activation. sHA significantly inhibited LNAI tumor growth. The majority of the animals did not form palpable tumors at 50-mg/kg dose even after 70 days when the treatment was stopped on 42nd day. No weight loss or serum and organ toxicity was observed in sHA treated animals. Tumors showed reduced microvessel density (⬃3-fold) and increased TUNEL positive cells (⬎ 5-fold). CONCLUSIONS: This is the first study that shows targeting HYAL1 (by sHA-a novel non-toxic agent), may control CaP growth and progression. Source of Funding: R01 CA 123063-04 (VBL); R01 CA 7282111 (VBL)

1282 TARGET SPECIFIC AND DYE ENCAPSULATED MICELLE PROBES FOR THE DETECTION OF EXTRA-PROSTATE CANCER DURING ROBOTIC-ASSISTED LAPAROSCOPIC PROSTATECTOMY (RALP) William K. Johnston III*, Evanston, IL; Jiufeng Fan, Liaohai Chen, Argonne, IL INTRODUCTION AND OBJECTIVES: During RALP, intra-operative determination of extra-capsular prostatic cancer growth is difficult and is reflected in the significant positive surgical margin rate of many surgical series. It is highly desired to develop efficient imaging regents that can clearly mark extra-prostatic cancer. We have previously reported our in vitro animal studies with fluorescein bound to PSMA antibody that demonstrated prostatic specific illumination. We sought to increase the fluorescent intensity by increasing the concentration of fluorescein bound to each ligand. METHODS: A novel fluorescent polymeric micelle probe was developed by encapsulating fluorescence molecules, with a higher self-quenching concentration threshold, within a micelle core while conjugating folic acid or prostate-specific membrane antigen (PSMA) inhibitor analog molecules on the surface of micelle. Fresh prostate tissue samples after RALP were obtained using a biopsy gun (⬃ 1 ⫻ 5 mm). Tissue samples were incubated with our fluorescent polymeric micelle probe for 30 minutes, followed by multiple washing and rinsing steps, and imaged under a fluorescence microscope (quantitated with Imaging j software). RESULTS: The ligand to dye ratio within each micelle was estimated to be 1:10,000. Given the higher self-quenching concentration threshold of the dye molecules, our polymeric micelle probe was much brighter compared to the conventional dye labeled antibody. When the prostate tissues were labeled with fluorescent polymeric micelles, the corresponding fluorescence intensity was 5⫻ stronger over the tissue autofluorescence. Tissues from peripheral zone compared to the median lobe exhibit 20% additional fluorescence intensity. Systematic blocking experiments were performed to demonstrate tissue specific properties of the folic acid/PSMA inhibitor conjugated fluorescent micelles. Up to 40% decrease of fluorescence intensity was observed when the tissues were blocked by high concentration of free folic acid or PSMA inhibitor.

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CONCLUSIONS: A new fluorescence probe locked within a micelle improved fluorescent intensity of prostate tissue. Since fluorescein dye, block copolymer, and the targeting ligand are FDA approved components, phase 0 and phase I clinic trials can be feasible once we address the effectiveness and toxicity issues in animal studies. The ability to identify extra-prostatic cancer could improve oncologic outcomes while decreasing morbidity during RALP. Source of Funding: Private Grant: NorthShore University

1283 F-BOX PROTEIN 10, AN NF-KB-DEPENDENT ANTI-APOPTOTIC PROTEIN, REGULATES TRAIL-INDUCED APOPTOSIS THROUGH MODULATING C-FOS/C-FLIP PATHWAY Rongbin Ge, Zongwei Wang, Qing Zeng, Xiaoyin Xu, Aria Olumi*, Boston, MA INTRODUCTION AND OBJECTIVES: Tumor necrosis factorrelated apoptosis-inducing ligand (TRAIL) holds great promise as a potential anti-cancer agent, but some tumors develop resistance to TRAIL. AP-1 family member, c-Fos, is an important modulator of apoptosis. Although F-box protein 10 (FBXL10) has been implicated to regulate an AP-1 family protein, significance and mechanism of FBXL10 in apoptosis has not been investigated. METHODS: TRAIL-sensitive (A498, PC3, MBA-MD-231) and TRAIL-resistant cells (LNCaP, PC3TR) were used, and a quantitative analysis of apoptosis was performed by using MTS and caspase-3, PARP activity as¬say and flow cytometry assay of annexin V- and propidium iodide-stained cells. RNAi, Chromatin immunoprecipitation (ChIP), EMSA, hydrodynamic-based transfection, oligonucleotide decoy technologies were performed to analyze the molecular interaction between FBXL10, c-Fos and NF-kB. The anti-apoptotic role of FBXL10 in vivo was analyzed by intravenous treatment with the TRAIL agonist compound, Lexatumumab. RESULTS: FBXL10 was suppressed and c-Fos was upregulated in TRAIL sensitive cancer cells and xenografts after treatment with TRAIL. However, in TRAIL resistant cancer cells and xenografts, FBXL10 and c-Fos were not affected. Expression of FBXL11, a FBXL10 homologous protein and c-Jun, was not affected by TRAIL. Silencing of FBXL10 sensitized resistant cells to TRAIL. Conversely, over-expression of FBXL10 repressed TRAIL-induced apoptosis. To behave as an anti-apoptotic molecule, FBXL10 was directly bound to the c-Fos promoter and repressed c-Fos levels in vitro and in vivo. In addition, FBXL10 regulated c-FLIP, another anti⫺apoptotic molecule, by a c-Fos dependent pathway. We also found that expression of FBXL10 is NF-kB-dependent, and TRAIL or proteasome inhibitors down-regulated FBXL10 via inhibiting NF-kB signaling. Using ChIP, EMSA assays and oligonucleotide decoy technologies, we found that NF-kB -p65 directly binds the FBXL10 promoter in vivo and in vitro, and promotes expression of FBXL10. CONCLUSIONS: Differentiating molecular mechanisms between TRAIL sensitive and resistant cancer cells will improve the efficacy of apoptotic targeted therapies for cancer patients. In this study, we demonstrate a novel functional role for FBXL10 as an anti-apoptotic molecule, and describe a new apoptotic mediated pathway that involves NF-kB/FBXL10/c-Fos/c-FLIP. Therefore, silencing FBXL10 can help overcome resistant cancer cells for pro-apoptotic therapies. Source of Funding: New York Academy of Medicine