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-κB (NF-κB) and signal transducer and activator of
transcription factor 3 (Stat3) activation and interaction have 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 GBM still remain unknown. Stat3 and NF-κB (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-κB
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-κB (+399) and Stat3 (+479) binding motifs in the proximal intron-1 in elevating IR-induced
ICAM-1 expression. Specific inhibition of Stat3 or 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 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-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.