Evasion of apoptosis can be caused by epigenetic silencing of caspase-8, a key component of the extrinsic apoptosis pathway. Loss of caspase-8 correlates with poor prognosis in medulloblastoma, which highlights the relevance of strategies to upregulate caspase-8 to break apoptosis resistance. Here, we develop a new combinatorial approach, that is treatment using histone deacetylase inhibitors (HDACI) together with interferon (IFN)-c, to restore caspase-8 expression and to overcome resistance to the death-receptor ligand TNF-related apoptosis-inducing ligand (TRAIL) in medulloblastoma in vitro and in vivo. HDACI, for example, valproic acid (VA), suberoylanilide hydroxamic acid (SAHA) and MS-275, cooperate with IFN-c to upregulate caspase-8 in cancer cells lacking caspase-8, thereby restoring sensitivity to TRAILinduced apoptosis. Molecular studies show that VA promotes histone acetylation and acts in concert with IFN-c to stimulate caspase-8 promoter activity. The resulting increase in caspase-8 mRNA and protein expression leads to enhanced TRAILinduced activation of caspase-8 at the death-inducing signaling complex, mitochondrial outer-membrane permeabilization and caspase-dependent cell death. Intriguingly, pharmacological or genetic inhibition of caspase-8 also abolishes the VA/IFN-cmediated sensitization for TRAIL-induced apoptosis. It is important to note that VA and IFN-c restore caspase-8 expression and sensitivity to TRAIL in primary medulloblastoma samples and significantly potentiate TRAIL-mediated suppression of medulloblastoma growth in vivo. These findings provide the rationale for further (pre)clinical evaluation of VA and IFN-c to restore caspase-8 expression and apoptosis sensitivity in cancers with caspase-8 silencing and open new perspectives to overcome TRAIL resistance.

Messenger RNAs are transported to the cytoplasm bound to several shuttling mRNA-binding proteins. Here, we present the characterization of Hrb1, a novel component of the transported ribonucleoprotein complex in Saccharomyces cerevisiae. The protein is similar to the other two serine/arginine (SR)-type proteins in yeast, Gbp2 and Npl3. Hrb1 is nuclear at steady state and its import is mediated by the karyopherin Mtr10. Hrb1 binds to poly(A)؉ RNA in vivo and its binding is significantly increased in MTR10 mutants, suggesting a role for Mtr10 in dissociating Hrb1 from the mRNAs. Interestingly, by comparing the export requirements of all three SR proteins we find similarities but also striking differences. While the export of all three proteins is dependent on the export of mRNAs in general, as no transport is observed in mutants defective in transcription (rpb1-1) or mRNA export (mex67-5), we find specific requirements for components of the THO complex, involved in transcription elongation. While both Hrb1 and Gbp2 depend on Mft1 and Hpr1 for their nuclear export, Npl3 is exported independently of both proteins. These findings suggest that Hrb1 and Gbp2, but not Npl3, might be loaded onto the growing mRNA via the THO complex components Mtf1 and Hrp1.

Apoptosis resistance is crucially involved in cancer development and progression, represents the leading cause for failure of anticancer therapy and is caused, for example, by downregulation of proapoptotic intracellular signaling molecules such as caspase-8. We found that the cytotoxic drugs methotrexate (MTX) and 5-fluorouracil (5-FU) were both able to sensitize resistant tumor cells for induction of apoptosis by p53-mediated upregulation of caspase-8. Increase in caspase-8 messenger RNA and protein expression disabled tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced proliferation and restored sensitivity toward TRAIL-induced apoptosis which was inhibited by transfection of p53 decoy oligonucleotides, p53 shRNA and caspase-8 shRNA. Upregulation of caspase-8 and sensitization toward TRAIL-induced apoptosis was found both in a broad panel of tumor cell lines with downregulated caspase-8 and in TRAIL-resistant primary tumor cells of children with acute leukemia. Taken together, we have identified caspase-8 as an important p53 target gene regulated by cytotoxic drugs. These findings highlight a new drug-induced modulation of physiological apoptosis pathways, which may be involved in successful anticancer therapy using MTX and 5-FU in leukemia and solid tumors over decades.

NF-κB is activated by DNA-damaging anticancer drugs as part of the cellular stress response. However, the consequences of drug-induced NF-κB activation are still only partly understood. To investigate the impact of NF-κB on the cell’s response to DNA damage, we engineered glioblastoma cells that stably express mutant IκBα superrepressor (IκBα-SR) to block NF-κB activation. Here, we identify a novel pro-apoptotic function of NF-κB in the DNA damage response in glioblastoma cells. Chemotherapeutic drugs that intercalate into DNA and inhibit topoisomerase II such as Doxorubicin, Daunorubicin and Mitoxantrone stimulate NF-κB DNA binding and transcriptional activity prior to induction of cell death. Importantly, specific inhibition of drug-induced NF-κB activation by IκBα-SR or RNA interference against p65 significantly reduces apoptosis upon treatment with Doxorubicin, Daunorubicin or Mitoxantrone. NF-κB exerts this pro-apoptotic function especially after pulse drug exposure as compared to continuous treatment indicating that the contribution of NF-κB becomes relevant during the recovery phase following the initial DNA damage. Mechanistic studies show that NF-κB inhibition does not alter Doxorubicin uptake and efflux or cell cycle alterations. Genetic silencing of p53 by RNA interference reveals that NF-κB promotes drug-induced apoptosis in a p53-independent manner. Intriguingly, drug-mediated NF-κB activation results in a significant increase in DNA damage prior to the induction of apoptosis. By demonstrating that NF-κB promotes DNA damage formation and apoptosis upon pulse treatment with DNA intercalators, our findings provide novel insights into the control of the DNA damage response by NF-κB in glioblastoma.

Epigenetic alterations are a hallmark of cancer that govern the silencing of genes. Up to now, 5-azacytidine (5-aza-CR, Vidaza) and 5-aza-2 0 -deoxycytidine (5-aza-dC, Dacogen) are the only clinically approved DNA methyltransferase inhibitors (DNMTi). Current effort tries to exploit DNMTi application beyond acute leukemia or myelodysplastic syndrome, especially to solid tumors. Although both drugs only differ by a minimal structural difference, they trigger distinct molecular mechanisms that are highly relevant for a rational choice of new combination therapies. Therefore, we investigated cell death pathways in vitro in human hepatoma, colon, renal, and lung cancer cells and in vivo in chorioallantoic membrane and xenograft models. Real-time cancer cell monitoring and cytokine profiling revealed a profoundly distinct response pattern to both drugs. 5-aza-dC induced p53-dependent tumor cell senescence and a high number of DNA double-strand breaks. In contrast, 5-aza-CR downregulated p53, induced caspase activation and apoptosis. These individual response patterns of tumor cells could be verified in vivo in chorioallantoic membrane assays and in a hepatoma xenograft model. Although 5-aza-CR and 5-aza-dC are viewed as drugs with similar therapeutic activity, they induce a diverse molecular response in tumor cells. These findings together with other reported differences enable and facilitate a rational design of new combination strategies to further exploit the epigenetic mode of action of these two drugs in different areas of clinical oncology. Mol Cancer Ther; 12(10); 2226-36. Ó2013 AACR.