NF-κB mediated ERK inhibition in radiation-induced radioresistance

4419 Transcription factors NF-κB and ERK are co-activated in acute response to many stress conditions. Their relationship in signaling the resistance of heterogeneous tumor cells to therapeutic radiation remains to be elucidated. The present study presents the evidence indicating an opposite regulation of NF-κB and ERK pathways in radioresistant breast cancer population (MCF+FIR) derived from fractionated doses of radiation. MCF+FIR cells displayed a heterogenic but temporary advantage of clonogenic survival with an increased NF-κB transcriptional activity and p65 and p50 (NF-κB subunits) expression. Unlike in single dose response, the level of phosphorylated ERK (pERK) was remarkably inhibited in all MCF+FIR cell lines with enhanced NF-κB activity. Phosphorylated MEK (pMEK) that activate ERK was inhibited in MCF+FIR cells but demonstrated a substantial level in MCF-7 cells. Both pMEK and pERK were inhibited with enhanced radioresistance by further irradiation with fractionated doses to MCF+FIR and control MCF-7 cells. Thus, MEK/ERK pathway is significantly down-regulated in radiation derived radioresistant cells. NF-κB subunit p65 interacts with MEK in MCF+FIR cells, suggesting an inhibitory role in MEK/ERK pathway via NF-κB. To identify the elements of up-stream of NF-κB, we observed an in vivo association of ATM and NF-κB in MCF+FIR cells but not in wild-type MCF-7 cells, indicating that ATM is involved in NF-kB activation. Direct protein interaction between p65 and ERK1 or ERK2 was further visualized in living cells by bimolecular fluorescence complementation using fusion proteins. p65/ERK interaction and nuclear translocation was significantly decreased in MCF+FIR cells and pMEK/pERK inhibitor PD98059 reduced p65/ERK interaction. In summary, these results demonstrate a novel communication between NF-κB and pMEK/pERK pathway in long-term radiation derived radioresistant populations.