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Identification of a novel pro-apopotic function of NF-kappaB in the DNA damage response.

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

SUBMITTER: Karl S

PROVIDER: S-EPMC4496130 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Identification of a novel pro-apopotic function of NF-kappaB in the DNA damage response.

Karl Sabine S Pritschow Yvonne Y Volcic Meta M Häcker Sabine S Baumann Bernd B Wiesmüller Lisa L Debatin Klaus-Michael KM Fulda Simone S

Journal of cellular and molecular medicine 20090901 10

NF-kappaB is activated by DNA-damaging anticancer drugs as part of the cellular stress response. However, the consequences of drug-induced NF-kappaB activation are still only partly understood. To investigate the impact of NF-kappaB on the cell's response to DNA damage, we engineered glioblastoma cells that stably express mutant IkappaBalpha superrepressor (IkappaBalpha-SR) to block NF-kappaB activation. Here, we identify a novel pro-apoptotic function of NF-kappaB in the DNA damage response in ...[more]

PMID: 19725919

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Project description:The ability of the transcription factor NF-kappaB to upregulate anti-apoptotic proteins has been linked to the chemoresistance of solid tumors to standard chemotherapy. In contrast, recent studies have proposed that, in response to doxorubicin, NF-kappaB can be pro-apoptotic through repression of anti-apoptotic target genes. However, there is little evidence analyzing the outcome of NF-kappaB inhibition on the cytotoxicity of doxorubicin in studies describing pro-apoptotic NF-kappaB activity. In this study, we further characterize the activation of NF-kappaB in response to doxorubicin and evaluate its role in chemotherapy-induced cell death in sarcoma cells where NF-kappaB is reported to be pro-apoptotic. Doxorubicin treatment in U2OS cells induced canonical NF-kappaB activity as evidenced by increased nuclear accumulation of phosphorylated p65 at serine 536 and increased DNA-binding activity. Co-treatment with a small molecule IKKbeta inhibitor, Compound A, abrogated this response. RT-PCR evaluation of anti-apoptotic gene expression revealed that doxorubicin-induced transcription of cIAP2 was inhibited by Compound A, while doxorubicin-induced repression of other anti-apoptotic genes was unaffected by Compound A or siRNA to p65. Furthermore, the combination of doxorubicin and canonical NF-kappaB inhibition with Compound A or siRNA to p65 resulted in decreased cell viability measured by trypan blue staining and MTS assay and increased apoptosis measured by cleaved poly (ADP-ribose) polymerase and cleaved caspase 3 when compared to doxorubicin alone. Our results demonstrate that doxorubicin-induced canonical NF-kappaB activity associated with phosphorylated p65 is anti-apoptotic in its function and that doxorubicin-induced repression of anti-apoptotic genes occurs independent of p65. Therefore, combination therapies incorporating NF-kappaB inhibitors together with standard chemotherapies remains a viable method to improve the clinical outcomes in patients with advanced stage malignancies.

Project description:BackgroundRearrangements involving the fibroblast growth factor receptor 1 (FGFR1) gene result in 8p11 myeloproliferative syndrome (EMS), which is a rare and aggressive hematological malignancy that is often initially diagnosed as myelodysplastic syndrome (MDS). Clinical outcomes are typically poor due to relative resistance to tyrosine kinase inhibitors (TKIs) and rapid transformation to acute leukemia. Deciphering the transcriptomic signature of FGFR1 fusions may open new treatment strategies for FGFR1 rearrangement patients.MethodsDNA sequencing (DNA-seq) was performed for 20 MDS patients and whole exome sequencing (WES) was performed for one HOOK3-FGFR1 fusion positive patient. RNA sequencing (RNA-seq) was performed for 20 MDS patients and 8 healthy donors. Fusion genes were detected using the STAR-Fusion tool. Fluorescence in situ hybridization (FISH), quantitative real-time PCR (qRT-PCR), and Sanger sequencing were used to confirm the HOOK3-FGFR1 fusion gene. The phosphorylation antibody array was performed to validate the activation of nuclear factor-kappaB (NF-kappaB) signaling.ResultsWe identified frequently recurrent mutations of ASXL1 and U2AF1 in the MDS cohort, which is consistent with previous reports. We also identified a novel in-frame HOOK3-FGFR1 fusion gene in one MDS case with abnormal monoclonal B-cell lymphocytosis and ring chromosome 8. FISH analysis detected the FGFR1 break-apart signal in myeloid blasts only. qRT-PCR and Sanger sequencing confirmed the HOOK3-FGFR1 fusion transcript with breakpoints located at the 11th exon of HOOK3 and 10th exon of FGFR1, and Western blot detected the chimeric HOOK3-FGFR1 fusion protein that is presumed to retain the entire tyrosine kinase domain of FGFR1. The transcriptional feature of HOOK3-FGFR1 fusion was characterized by the significant enrichment of the NF-kappaB pathway by comparing the expression profiling of FGFR1 fusion positive MDS with 8 healthy donors and FGFR1 fusion negative MDS patients. Further validation by phosphorylation antibody array also showed NF-kappaB activation, as evidenced by increased phosphorylation of p65 (Ser 536) and of IKBalpha (Ser 32).ConclusionsThe HOOK3-FGFR1 fusion gene may contribute to the pathogenesis of MDS and activate the NF-kappaB pathway. These findings highlight a potential novel approach for combination therapy for FGFR1 rearrangement patients.

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Identification of a novel pro-apopotic function of NF-kappaB in the DNA damage response.

Publications

Identification of a novel pro-apopotic function of NF-kappaB in the DNA damage response.

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