960. Forced Expression of the Heat Shock Protein H11 Causes Growth Arrest and Apoptosis of Melanoma Cells through TAK1 Activation and Inhibits Tumor Growth in Xenograft Models

CANCER-TARGETED GENE THERAPY: OTHER VIRUSES AND NEW APPROACHES 959. Tumor Therapy Via Vector-Regulated Expression of Mutant Sodium Channel Bakhos A. Tannous,1,2,3 Katherine F. Perry,2 Miguel SenaEsteves,2 Jaime Garcias-Anoveros,4 Andreas Jacobs,5 David P. Corey,6 Ralph Weissleder,1 Xandra O. Breakefield.1,2,3 1 Center for Molecular Imaging Research, Massachusetts General Hospital, Boston, MA; 2Molecular Neurogenetics Unit, Massachusetts General Hospital, Boston, MA; 3Neuroscicence Program, Harvard Medical School, Boston, MA; 4Department of Anesthesiology, Northwestern University Feinberg School of Medicine, Chicago, IL; 5Neurology, University of Koln, Koln, Germany; 6Howard Hughes Medical Institute and Neurobiology, Harvard Medical School, Boston, MA. Viral vectors provide a means to deliver therapeutic genes to tumors on site. In this study, we describe a novel tumor therapy employing a dominant-negative mutant form of a mammalian brain sodium channel, BNac1. This channel was delivered to tumor cells using an HSV amplicon vector in which gene expression is controlled by a tetracycline regulatory system (tet-on) with silencer elements. Channel activity, regulated transgene expression, cell death and the bystander effect were evaluated in glioma cells in culture. Upon infection and drug induction, the constitutively open channel is expressed leading to sodium and water influx into cells with swelling and death (Fig. 1). Gap junctions were demonstrated between tumor cells and mediated a bystander effect by ion influx whereby noninfected tumor cells adjacent to infected tumor cells were also killed. Growth and therapeutic response in culture and in vivo were monitored, in real-time, in experimental tumors expressing a recently characterized Gaussia luciferase that is far more sensitive than other luciferases currently in use.

960. Forced Expression of the Heat Shock Protein H11 Causes Growth Arrest and Apoptosis of Melanoma Cells through TAK1 Activation and Inhibits Tumor Growth in Xenograft Models Baiquan Li,1 Cynthia C. Smith,1 Jennifer M. Laing,1 Laure Aurelian.1 1 Pharmacology and Experimental Therapuetics, University of Maryland, Baltimore, Baltimore, MD. The newly cloned small heat shock protein H11 evidences celltype specific silencing by aberrant DNA methylation in 5/9 melanoma cell lines. In these cells, the de-methylating agent Aza-C causes H11 overload and triggers growth arrest and apoptosis. Apoptosis is blocked by antisense, but not sense, H11 oligonucleotides, indicating that it is induced by H11. Aza-C treatment did not induce H11 expression nor trigger growth arrest/apoptosis in cultured normal melanocytes. To identify the mechanism of growth arrest/apoptosis induced by H11 overload, melanoma cells were stably transfected with H11 under the control of a tetracycline sensitive promoter and examined for altered gene expression and cell survival before and after treatment with Doxycycline (Dox). Dox (2 µg/ml) caused a time-dependent increase in H11 expression, reaching maximal levels on day 3 post treatment. H11 overload was accompanied by growth arrest (first seen on days 1-2 post Dox), activation of caspases-9 and3, seen as early as day 1 post Dox and a time-dependent increase in the

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% TUNEL+ (apoptotic) cells (70-85% on days 3-4 post Dox). Immunoprecipitation/immunoblotting and immunocomplex PK assays indicated that H11 bound and activated TAK1. TAK1 did not bind and was not activated by the H11 mutant, W51C which is dominant negative for apoptosis. Caspase activation was inhibited with the dominant negative TAK1 mutant K63W or the p38MAPK pharmacologic inhibitor SB203580, indicating that apoptosis is caused by H11-mediated activation of the TAK1/p38MAPK pathway. H11-mediated growth arrest was also caused by activated TAK1, through phosphorylation of b-catenin and inhibition of MITF and CDK2, which are required for melanoma cell proliferation. H11 did not bind nor activate TAK1 in normal melanocytes. Studies of the highly tumorigenic A2058 and LM melanoma cultures in a mouse xenograft model, indicated that tumor growth was significantly (p<0.001) reduced by treatment with Aza-C (5mg/kg) given by ip injection on days 6 and 15 after initiation of the xenografts (3 doses at 3hr intervals). Decreased tumor growth was due to H11 induced apoptosis as evidenced by H11/TUNEL co-localization in residual tumor cells. The data suggest that H11 is a promising target for chemogene therapy of melanoma and identify additional members of the H11-mediated pro-apoptotic cascade.

961. Angiopoietin-1 Enhances the Effect of Paclitaxel on Breast Cancer Cell Killing in a Mouse Model YaoQi Huang,1 Harald Sauthoff,1,2 Jin Cheng,1 David Kutler,2,3 Sheila Heitner,1 William N. Rom,1 John G. Hay.1,2 1 Medicine, NYU School of Medicine, New York, NY; 2Medicine, Veterans Administration, New York, NY; 3Otolaryngology, NYU School of Medicine, New York, NY. Angiogenesis has an important role in tumor growth and metastasis. Various factors, such as VEGFs and angiopoietins, have been implicated in the regulation of vessel formation. Angiopoietins regulate angiogenesis by activating or blocking the activation of Tie2, a surface receptor tyrosine kinase generally restricted to endothelial cells. Angiopoietin-1 (Ang1) promotes endothelial cell survival, helps to maintain vascular integrity and is thus involved in vessel maturation. Conversely, Ang2 acts as an antagonist of the Ang1/ Tie2 interaction. Solid tumors require blood vessels for growth, although tumor neovascularization is structurally and functionally abnormal with leaky and tortuous vessels. These abnormalities contribute to impaired blood supply and interstitial hypertension, which interferes with the delivery of therapeutics to the tumor. Since Ang1 stabilizes blood vessels and inhibits vascular permeability, we hypothesized that normalization of tumor vasculature by Ang1 could improve drug delivery and thereby enhance cancer cell killing. The impact of Ang1 delivered by a non-replicating adenoviral vector (AdAng1) on the anti-tumor effect of paclitaxel was studied in a 4T1 murine breast cancer model. The expression and secretion of Ang1 from 4T1 cells infected by AdAng1 in vitro was first confirmed by immunostaining and immunoblotting. AdAng1 did not affect the growth of 4T1 cells in vitro, as the growth rates of 4T1 cells infected with AdAng1 or control AdCon virus were similar. Paclitaxel did inhibit the growth of 4T1 cells in vitro, but the growth inhibition was similar in control virus and AdAng1 treated cultures. For the in vivo model, 4T1 cells were first treated with either AdAng1, AdCon or PBS, and then injected into the flank of syngeneic BALB/c mice. Paclitaxel was administered by i.p. injection twice a week, starting at day 7. The combination of AdAng1 administration together with Paclitaxel reduced tumor growth compared to Paclitaxel alone, AdAng1 alone or control virus plus Paclitaxel. In summary, this study indicates that Ang1 can enhance the effect of chemotherapy in a breast cancer model. These effects are likely to be related to Ang1-mediated vessel normalization and thereby improved drug delivery to the tumor cells. Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright  The American Society of Gene Therapy