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

Abstract

The newly cloned small heat shock protein H11 evidences cell-type 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 |[mu]|g/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 and-3, seen as early as day 1 post Dox and a time-dependent increase in the% 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.