NF-κB is involved in immune responses, inflammation, oncogenesis, cell proliferation and apoptosis. Even though NF-κB can be activated by DNA damage via Ataxia telangiectasia-mutated (ATM) signalling, little was known about an involvement in DNA repair. In this work, we dissected distinct DNA double-strand break (DSB) repair mechanisms revealing a stimulatory role of NF-κB in homologous recombination (HR). This effect was independent of chromatin context, cell cycle distribution or cross-talk with p53. It was not mediated by the transcriptional NF-κB targets Bcl2, BAX or Ku70, known for their dual roles in apoptosis and DSB repair. A contribution by Bcl-xL was abrogated when caspases were inhibited. Notably, HR induction by NF-κB required the targets ATM and BRCA2. Additionally, we provide evidence that NF-κB interacts with CtIP–BRCA1 complexes and promotes BRCA1 stabilization, and thereby contributes to HR induction. Immunofluorescence analysis revealed accelerated formation of replication protein A (RPA) and Rad51 foci upon NF-κB activation indicating HR stimulation through DSB resection by the interacting CtIP–BRCA1 complex and Rad51 filament formation. Taken together, these results define multiple NF-κB-dependent mechanisms regulating HR induction, and thereby providing a novel intriguing explanation for both NF-κB-mediated resistance to chemo- and radiotherapies as well as for the sensitization by pharmaceutical intervention of NF-κB activation.

Inhibitor of apoptosis (IAP) proteins are expressed at high levels in many cancers and therefore represent attractive targets for therapeutic intervention. Here, we report for the first time that the second mitochondria-derived activator of caspases (Smac) mimetic BV6 sensitizes glioblastoma cells toward Temozolomide (TMZ), the first-line chemotherapeutic agent in the treatment of glioblastoma. BV6 and TMZ synergistically reduce cell viability and trigger apoptosis in glioblastoma cells (combination index o0.4-0.8), which is accompanied by increased loss of mitochondrial-membrane potential, cytochrome c release, caspase activation and caspase-dependent apoptosis. Analysis of the molecular mechanisms reveals that BV6 causes rapid degradation of cIAP1, leading to stabilization of NF-kB-inducing kinase and NF-kB activation. BV6-stimulated NF-kB activation is critically required for sensitization toward TMZ, as inhibition of NF-kB by overexpression of the mutant IkBa super-repressor profoundly reduces loss of mitochondrial membrane potential, cytochrome c release, caspase activation and apoptosis. Of note, BV6mediated sensitization to TMZ is not associated with increased tumor necrosis factor alpha (TNFa) production. Also, TNFa, CD95 or TRAIL-blocking antibodies or knockdown of TNFR1 have no or little effect on combination treatment-induced apoptosis. Interestingly, BV6 and TMZ cooperate to trigger the formation of a RIP1 (receptor activating protein 1)/caspase-8/FADD complex. Knockdown of RIP1 by small interfering RNA significantly reduces BV6-and TMZ-induced caspase-8 activation and apoptosis, showing that RIP1 is necessary for apoptosis induction. By demonstrating that BV6 primes glioblastoma cells for TMZ in a NF-kB-and RIP1-dependent manner, these findings build the rationale for further (pre)clinical development of Smac mimetics in combination with TMZ.

Evasion of apoptosis contributes to radioresistance of glioblastoma, calling for novel strategies to overcome apoptosis resistance. In this study, we investigated the potential of the small molecule Smac mimetic BV6 to modulate radiosensitivity of glioblastoma cells. Here, we identify a novel proapoptotic function of NF-kB in g-irradiation-induced apoptosis of glioblastoma cells by showing, for the first time, that NF-kB is critically required for Smac mimetic-mediated radiosensitization. BV6 significantly increases g-irradiation-triggered apoptosis in several glioblastoma cell lines in a dose-and time-dependent manner. Calculation of combination index (CI) reveals that the interaction of BV6 and g-irradiation is highly synergistic (CI < 0.3). Molecular studies show that BV6 stimulates NF-kB activation, which is critical for radiosensitization, because genetic inhibition of NF-kB by overexpression of the dominant-negative superrepressor IkBa-SR significantly decreases BV6-and g-irradiation-induced apoptosis. Also, the BV6-mediated enhancement of g-irradiation-triggered caspase activation, drop of mitochondrial membrane potential, and cytochrome c release is abolished in cells overexpressing IkBa-SR. Similarly, NF-kB inhibition by ectopic expression of a kinase dead mutant of IKKb prevents the BV6-mediated sensitization for g-irradiation. The clinical relevance is underscored by experiments with primary tumor samples showing that BV6 sensitizes primary cultured glioma cells as well as glioblastoma-initiating cancer stem cells derived from surgical specimens for g-irradiation. In conclusion, we identify NF-kB as a critical mediator of Smac mimetic-conferred radiosensitization of glioblastoma cells. These results have important implications for the development of Smac mimetic-based combination protocols for radiosensitization of glioblastoma. Mol Cancer Ther; 10(10); 1867-75. Ó2011 AACR.

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.

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Despite aggressive therapies, the prognosis of children with high-risk medulloblastoma is still poor, thus underscoring the need to develop novel treatment strategies. Here, we report that histone deacetylase inhibitors (HDACI), that is, MS-275, valproic acid or SAHA, provide a novel strategy for sensitization of medulloblastoma to DNA-damaging drugs such as Doxorubicin, VP16 and Cisplatin by promoting p53-dependent, mitochondrial apoptosis. Mechanistic studies reveal that single-agent treatment with MS-275 causes acetylation of the non-histone protein Ku70, an event reported to release Bax from Ku70, whereas DNAdamaging drugs trigger p53 acetylation and accumulation. Combined treatment with MS-275 and Doxorubicin or VP16 cooperates to promote binding of p53 to Bax and p53-dependent Bax activation, resulting in enhanced loss of mitochondrial membrane potential, cytochrome c release and caspase-dependent apoptosis. Overexpression of Bcl-2 almost completely abolishes the MS-275-mediated chemosensitization, underlining the importance of the mitochondrial pathway for inducing apoptosis. Also, MS-275 cooperates with chemotherapeutics to inhibit long-term clonogenic survival. Most importantly, MS-275 increases chemotherapeutic drug-induced apoptosis in primary medulloblastoma samples, and cooperates with Doxorubicin to suppress medulloblastoma growth in an in vivo model, which underscores the clinical relevance of the findings. Thus, HDACI such as MS-275 present a promising approach for chemosensitization of medulloblastoma by enhancing mitochondrial apoptosis in a p53-dependent manner. These findings have important clinical implications for the design of experimental treatment protocols for medulloblastoma.