Escaping apoptosis is a hallmark of cancer. Resistance to apoptosis can be caused by epigenetic silencing of caspase-8, which correlates with poor prognosis in medulloblastoma, the most common primary malignant brain tumor in childhood. In search for novel strategies to restore defective apoptosis in cancer cells, we investigated the role of chromatin remodeling in transcriptional regulation of caspase-8. Here, we identify aberrant histone deacetylation as a novel mechanism of epigenetic inactivation of caspase-8. Histone deacetylase inhibitors (HDACI), e.g. valproic acid (VA), SAHA or MS-275, induced caspase-8 expression in a variety of neuroblastoma or medulloblastoma cell lines lacking caspase-8. Importantly, combined treatment with VA and Interferon (IFN)-γ synergized to upregulate caspase-8 compared to each agent alone. Treatment with VA/IFNγ caused globally increased acetylation of histone H4 as well as enhanced histone acetylation at the caspase-8 promoter as determined by ChIP assay. This chromatin remodeling at the caspase-8 promoter triggered by VA/IFNγ resulted in elevated caspase-8 promoter activity and increased caspase-8 mRNA levels. Consequently, re-expression of caspase-8 by VA/IFNγ also restored caspase-8 function, i.e. caspase-8 enzymatic activity upon TRAIL stimulation. This increased caspase-8 activity in turn triggered full activation of effector caspases such as caspase-3. Also, restoration of caspase-8 by VA/IFNγ initiated a mitochondrial amplification loop upon TRAIL treatment by causing drop of mitochondrial membrane potential and cytochrome c release from mitochondria into the cytosol. Importantly, pre-treatment with VA/IFNγ significantly sensitized otherwise resistant neuroectodermal tumor cells to TRAIL-induced apoptosis in a caspase-dependent manner, since this sensitization effect was blocked by the pancaspase inhibitor zVAD.fmk. To test the specific contribution of caspase-8 for the apoptosis sensitization effect provided by VA/IFNγ, we knocked down caspase-8 by RNA interference (RNAi) and also used the caspase-8 specific inhibitor zIETD.fmk. Intriguingly, specific inhibition of caspase-8 upregulation in response to VA/IFNγ treatment by caspase-8 RNAi or blockade of caspase-8 activity by zIETD.fmk also completely abolished the sensitization effect of VA/IFNγ for TRAIL-induced apoptosis showing that caspase-8 was required for this sensitization effect. By demonstrating that HDACI, especially in combination with IFNγ, restore caspase-8 expression and sensitize otherwise resistant tumor cells for TRAIL-induced apoptosis, our findings have important clinical implications for the use of TRAIL in cancer therapy. Thus, HDACI present a novel strategy to overcome TRAIL resistance in caspase-8 negative tumors.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA