495 CHEMOPREVENTION OF TUMOR PROGRESSION AND METASTASIS IN PROSTATE CANCER BY NUTRACEUTICAL, 4-METHYLUMBELLIFERONE

Vol. 189, No. 4S, Supplement, Sunday, May 5, 2013

494 MESENCHYMAL STEM CELL MEDIATED CANCER THERAPY INHIBITS TUMOR GROWTH IN THE TRANSGENIC ADENOCARCINOMA OF THE MOUSE PROSTATE (TRAMP) MODEL Roberta Buono*, Alberto Abrate, Antonio Esposito, Tamara Canu, Alessandro Del Maschio, Fabio Benigni, Petter Hedlund, Francesco Montorsi, Ilaria T. R. Cavarretta, Milan, Italy INTRODUCTION AND OBJECTIVES: Vectors that home tumors and micrometastases may be a unique tool to activate drugs selectively within a cancer site. Mesenchymal stem cells (MSC) as vectors for the prodrug-activating enzyme cytosine deaminase::uracil phosphoribosyltransferase (CD) convert the nontoxic 5-fluorocytosine (5FC, prodrug) into the antitumor drug 5-fluorouracil. Conversion occurs within the tumor mass thanks to the innate ability of MSC to target tumor lesions. We previously demonstrated that CD-MSC are effective in inhibiting growth of prostate tumor grafts in mice. Our objective was to further evaluate this therapeutic strategy in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model that better mimics onset and progression of human prostate disease. METHODS: After ethical approval, human adipose tissue (AT) MSC and mouse bone marrow (BM) MSC were used as vectors for CD. TRAMP mice were employed as a preclinical prostate cancer model. Autochthonous tumor volume was measured by magnetic resonance imaging (MRI) performed on a 7T preclinical scanner with multiplanar high resolution T2-weighted. Images post-processing was performed on MIPAV 12.5 software. Two millions CD-AT-MSC (first treatment) and CD-BM-MSC (second treatment) were injected into the tail vein. Prodrug (300 mg/kg/day) was administered intraperitoneally for 7-10 days after delivery of therapeutic cells. Mann-Whitney U test was used for comparisons RESULTS: TRAMP mice (ca. 26 weeks old) were divided into the following groups: CONTROL (received CD-MSC alone or 5FC alone) and TREATED (received CD-MSC plus 5FC). In the CONTROL group not all animals reached the time of the second MRI (some mice were sacrificed before second MRI for ethical reasons). Before therapy median TV was 91.19 mm3 ⫾ 14.9 for CONTROL mice (n⫽6) and 106.25 mm3 ⫾ 19.24 for TREATED mice (n⫽7). After therapy median TV was 106.77 mm3 ⫾ 21.59 for CONTROL mice and 83.86 mm3 ⫾ 14.09 for TREATED mice. We could therefore observe a median TV increase by 16.18% for the CONTROL mice and a median TV decrease by 28.72% for the TREATED mice (p⬍0.05). CONCLUSIONS: Systemic administration of CD-AT-MSC and CD-BM-MSC and subsequent prodrug treatment inhibit autochthonous prostate tumor growth. If confirmed in future larger studies, specifically engineered MSC cells may offer a new strategy to improve efficacy and selectivity of current antitumor therapies. Source of Funding: unrestricted grant from SANOFI.

495 CHEMOPREVENTION OF TUMOR PROGRESSION AND METASTASIS IN PROSTATE CANCER BY NUTRACEUTICAL, 4-METHYLUMBELLIFERONE Nicolas Ortiz*, Travis Yates, Luis Lopez, Marie Hupe, Vinata Lokeshwar, Miami, FL INTRODUCTION AND OBJECTIVES: Hyaluronic acid (HA), a glycosaminoglycan, is associated with prostate cancer (PCa) progression and metastasis. 4-methylumbelliferone (4-MU), is a HA-synthesis inhibitor. We have shown antitumor and anti-invasive effects of 4-MU on PCa cells in vitro and in xenografts. In this study we evaluated the efficacy of 4-MU to prevent PCa growth and metastasis in the TRAMP model. We also evaluated effects of 4-MU on epithelial-mesenchymal transition (EMT). METHODS: TRAMP mice (n ⫽ 8-11/group) were treated with vehicle or 4-MU (450 mg/kg) by starting stage-specific treatment at 8 (PIN stage; Gr1), 12 (adenocarcinoma; Gr2) and 22 (invasive carci-

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noma; Gr3) wks of age. At 28 wks, 50% of animals in 4-MU treated groups were sacrificed and the other 50% were left untreated up to 52 wks. At necropsy, prostate (P) and seminal vesicles (SV) were weighed, tissues were analyzed by histology, immunohistochemistry and quantitative PCR for EMT markers and serum chemistry was evaluated. PC3-ML cells treated with 4-MU were analyzed for EMT markers. RESULTS: In the TRAMP model, at 28 wks, 100% of vehicle treated mice developed prostate tumors, but tumor growth and progression was inhibited in all 4-MU treated groups; P⫹SV weight (g): vehicle: 3.2⫾2.9; 4-MU treated: Gr1: 0.43⫾0.14; Gr2: 0.35⫾0.04; Gr3: 1.0⫾0.35 g. P⫹SV weight in control TRAMP mice at 22 wks of age was 0.9⫾0.04. Animals in Gr1 and Gr2 remained tumor free until 52 wk and 44 wk respectively. While 55% of vehicle treated animals developed metastasis, none of the treated groups had metastasis or SV invasion. No serum/organ toxicity or weight loss was observed in the treatment groups. HA, HA receptors (CD44, RHAMM) and EMT markers (␤catenin, Zeb2) were downregulated (⬎2-fold), but E-cadherin was upregulated (4-fold) in the treatment groups. 4-MU treatment (0-60 ␮g/ml) similarly modulated the expression of EMT markers and HA receptors in PC3-ML cells. CONCLUSIONS: 4-MU is a promising non-toxic, oral chemopreventive agent, which inhibits PCa growth and metastasis by plausibly abrogating HA-signaling and reversing EMT. Source of Funding: R01 CA 123063-04 (VBL); R01 CA 7282111 (VBL).

496 FIBROBLAST GROWTH FACTOR 9 IN PROSTATE CANCER CELLS IS ASSOCIATED WITH POSTOPERATIVE RECURRENCE THROUGH ACCELERATION OF MESENCHYMAL TRANSITION, PROLIFERATION, AND INVASION Jun Teishima*, Koichi Shoji, Tetsutaro Hayashi, Shigeki Yano, Kiyotaka Oka, Hirotaka Nagamatsu, Shinya Ohara, Akio Matsubara, Hiroshima, Japan INTRODUCTION AND OBJECTIVES: Disruption of the fibroblast growth factor (FGF) signaling pathway in the prostate has been reported to cause failure of tissue homeostasis and development of malignant disease. Among the FGF family, FGF9 enhances cell proliferation and invasiveness in several malignant diseases. The aim of the present study is to investigate the role of FGF9 in prostate cancer cells. METHODS: Cell viability and invasion of LNCaP and PC3 cells were assessed by using MTT assay and Matrigel invasion assay, respectively, by treatment with recombinant FGF9. Expression of MMPs, VEGFs, and cadherins in LNCaP by incubation with FGF9 was assessed by western blot analysis. Tissues obtained during a radical prostatectomy in 133 male patients were immunohistochemically stained by using anti-FGF9, anti-cadherin, anti-VEGF, and anti-MMP antibodies. RESULTS: Cell viability and invasion of LNCaP and PC3 cells were significantly enhanced by treatment with recombinant FGF9. These effects were significantly suppressed by treatment with antiFGF9 neutralizing antibody. Expression of N-cadherin, VEGF-A, and MMP2 were induced in LNCaP cells incubated in medium with FGF9 for five days. Immunohistochemical staining detected FGF9-positive cells in 20 samples. Furthermore, the immunoreactivity of N-cadherin and VEGF-A was highly detected in FGF9-positive samples. The prevalence of FGF9-positive cells was 34.2% in cases diagnosed with a Gleason score of 8 or higher (vs. Gleason score of 7 or lower, 7.4%, p⫽0.0003), 43.8% with seminal vesicle invasion (vs. without seminal vesicle invasion, 11.1%, p⫽0.0022), or 27.7% for those whose serum PSA level was higher than 10 ng/ml (vs. those whose serum PSA level was 10 ng/ml or lower, 8.1%, p⫽0.0058), significantly higher in comparison with other cases. The third-year biochemical relapse-free survival rate was 17.5% in cases with FGF9-positive cells, which was significantly lower than that in cases in which FGF9-positive cells were