The activation of NF-κB by neutrophil lactoferrin (Lf) is regulated via the IκB kinase (IKK) signaling cascade, resulting in the sequential phosphorylation and degradation of IκB. In this study, we observed that Lf protein augmented p65 phosphorylation at the Ser536, but not the Ser276 residue, and stimulated the translocation of p65 into the nucleus. Lf was also shown to enhance the association between p65 and CREB-binding protein/p300 in vivo. To elucidate the mechanism by which Lf triggers these signaling pathways, we attempted to delineate the roles of the upstream components of the IKK complex, using their dominant-negative mutants and IKKα−/− and IKKβ−/− mouse embryonic cells. We demonstrated that both IKKα and IKKβ as well as NF-κB-inducing kinase are indispensable for Lf-induced p65 phosphorylation. However, MAPK kinase kinase 1 is not essentially required for this activation. We also observed that Lf-induced p65 phosphorylation was either partially or completely abrogated as the result of treatment with the mutant forms of TNFR-associated factor (TRAF) 2, TRAF5, or TRAF6. Moreover, we demonstrated that Lf directly interacted with TRAF5. Expression of the dominant-negative mutant of TRAF5 or its small interfering RNA almost completely abrogated the Lf-induced p65 phosphorylation. These results suggest that signaling pathways, including TRAFs/NF-κB-inducing kinase/IKKs, may be involved in the regulation of Lf-induced p65 activation, thereby resulting in the activation of members of the NF-κB family.

Ionizing radiation (IR) is widely used as a main treatment for solid tumors, however, its potential influence and its role in the invasion and metastasis of cancer still remain unknown. In this study, we found that IR showed an inhibitory effect on the invasion of mutant p53 expressing cancer cells through targeting cyclin D1, which has been known as implicated in invasion and metastasis. Our data showed that gamma-irradiation (5 Gy) decreased trans-well migration of mutant p53 expressing cancer cells. It has been reported that following exposure to IR cyclin D1 is rapidly degraded via the ubiquitin pathway. We showed that IR-induced cyclin D1 degradation is restored after 24 hr in H1299 p53 negative cells. In contrast, cyclin D1 depletion was not recovered in H1299 mutant p53 cells, suggesting its inhibiting effect on cyclin D1 regulation. In addition, the levels of matrix metalloproteinase 9 were decreased in mutant p53 expressing cancer cells, indicating the reduction of cell migration and invasion. These data indicate that IR suppresses the restoration of cyclin D1 via the mutant p53, thus inhibiting the invasion of cancer cells expressing mutant p53. Citation Format: Shin-Hee Lee, Phillip Craigmile, Shiyong Wu. Ionizing radiation regulates mutant p53-mediated cancer cell invasiveness through targeting cyclin D1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 507. doi:10.1158/1538-7445.AM2015-507