Role of IKK and oscillatory NFkappaB kinetics in MMP-9 gene expression and chemoresistance to 5-fluorouracil in RKO...

Nuclear factor kappa B (NFkappaB) is a central participant in the metastasis and chemoresistance of colorectal cancer (CRC). However, it is not fully understood to what extent NFkappaB contributes to induction of the metastasis-associated matrix metalloprotease-9 (MMP-9) gene and sensitivity to the commonly used chemotherapeutic 5-fluorouracil (5-Fu) in CRC. Using the RKO human CRC cell line and two NFkappaB signaling deficient RKO mutants, we investigated NFkappaB's role in the induction of MMP-9 and 5-Fu sensitivity in RKO CRC cells. NFkappaB plays a predominant role in MMP-9 gene induction in RKO cells, as evidenced by the failure of tumor necrosis factor alpha (TNFalpha) to induce MMP-9 in either of the NFkappaB signaling mutants. RKO cells exhibit a robust, oscillatory NFkappaB activity in response to TNFalpha not seen in either of the NFkappaB mutant cell lines, which instead demonstrate diminished, nonoscillatory NFkappaB activation. Analysis of TNFalpha-induced phosphorylation and MMP-9 promoter recruitment of the p65 NFkappaB subunit revealed a significant reduction in p65 phosphorylation as well as reduced and altered recruitment of p65 to the MMP-9 gene promoter in the mutants compared to the parental RKO cell line. 5-Fu only activated NFkappaB in the parental RKO cells through induction of IkappaB-kinase (IKK) activity and increased sensitivity to 5-Fu is observed in both NFkappaB mutant lines. Our results suggest that TNFalpha-dependent induction of MMP-9 gene expression is tightly regulated by oscillatory/cumulative activation of NFkappaB and that 5-Fu stimulates NFkappaB and RKO CRC cell survival through induction of IKK activity.