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

摘要

Although radiotherapy improves survival in patients, GBMs tend to relapse with augmented tumor migration and invasion even after irradiation (IR). Aberrant NF-κB and Stat3 activation and interaction has been suggested in several human tumors. However, possible NF-κB/Stat3 interaction and the role of Stat3 in maintenance of NF-κB nuclear retention in glioblastoma (GBM) still remain unknown. Stat3 and NF-κB (p65) physically interact with one another in the nucleus in glioma tumors. Most importantly, GST pull-down assays identified that Stat3 binds to the p65 transactivation domain (TAD) and is present in the NF-κB DNA-binding complex. Irradiation significantly elevated nuclear phospho-p65/phospho-Stat3 interaction in correlation with increased ICAM-1 and sICAM-1 levels, migration and invasion in human glioma xenograft cell lines 4910 and 5310. ChIP and promoter luciferase activity assays confirmed the critical role of adjacent NF-κB (+399) and Stat3 (+479) binding motifs in the proximal intron-1 in elevating IR-induced ICAM-1 expression. Specific inhibition of Stat3 and NF-κB with Stat3.siRNA or JSH-23 severely inhibited IR-induced p65 recruitment onto ICAM-1 intron-1 and suppressed migratory properties in both 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-κB 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-κB/Stat3 nuclear association in IR-induced ICAM-1 regulation and implies that targeting NF-κB/Stat3 interaction may have future therapeutic significance in glioma treatment.