Essential role of cooperative NF-κB and Stat3 recruitment to ICAM-1 intronic consensus elements in the regulation of radiation-induced invasion and migration in glioma

Although radiotherapy improves survival in patients, glioblastoma multiformes (GBMs) tend to relapse with augmented tumor migration and invasion even after ionizing radiation (IR). Aberrant nuclear factor-kappa B (NF-kappa B) and signal transducer and activator of transcription factor 3 (Stat3) activation and interaction have been suggested in several human tumors. However, possible NF-kappa B/Stat3 interaction and the role of Stat3 in maintenance of NF-kappa B nuclear retention in GBM still remain unknown. Stat3 and NF-kappa B (p65) physically interact with one another in the nucleus in glioma tumors. Most importantly, glutathione S-transferase pull-down assays identified that Stat3 binds to the p65 transactivation domain and is present in the NF-kappa B DNA-binding complex. Irradiation significantly elevated nuclear phospho-p65/phospho-Stat3 interaction in correlation with increased intercellular adhesion molecule-1 (ICAM-1) and soluble-ICAM-1 levels, migration and invasion in human glioma xenograft cell lines 4910 and 5310. Chromatin immunopreicipitation and promoter luciferase activity assays confirmed the critical role of adjacent NF-kappa B (+399) and Stat3 (+479) binding motifs in the proximal intron-1 in elevating IR-induced ICAM-1 expression. Specific inhibition of Stat3 or NF-kappa B with Stat3. siRNA or JSH-23 severely inhibited IR-induced p65 recruitment onto ICAM-1 intron-1 and suppressed migratory properties in both the cell lines. On the other hand, Stat3C- or IR-induced Stat3 promoter recruitment was significantly decreased in p65-knockdown cells, thereby suggesting the reciprocal regulation between p65 and Stat3. We also observed a significant increase in NF-kappa B enrichment on ICAM-1 intron-1 and ICAM-1 transactivation in Stat3C overexpressing cells. In in vivo orthotopic experiments, suppression of tumor growth in Stat3.si + IR-treated mice was associated with the inhibition of IR-induced p-p65/p-Stat3 nuclear colocalization and ICAM-1 levels. To our knowledge, this is the first study showing the crucial role of NF-kappa B/Stat3 nuclear association in IR-induced ICAM-1 regulation and implies that targeting NF-kappa B/Stat3 interaction may have future therapeutic significance in glioma treatment.